Luminal grafts and methods of making and using the same

ABSTRACT

Luminal grafts and methods of making and using the same. An exemplary luminal graft of the present disclosure is configured as a generally tubular element configured for nerve cells to grow therethrough and comprises at least one sheet of biological tissue having elastin fibers and collagen fibers, with the elastin fibers being a dominant component thereof; and a plurality of microchannels formed on a surface of the at least one sheet of biological tissue, each of the microchannels extending longitudinally between a first end and a second end of the at least one sheet of biological tissue and configured to provide intraluminal structural guidance to nerve cells proliferating therethrough.

PRIORITY & RELATED APPLICATIONS

The present application is related to, claims the priority benefit of,and is a U.S. continuation patent application of, U.S. patentapplication Ser. No. 15/957,699, filed Apr. 19, 2018 and issued as U.S.Pat. No. 11,000,285 on May 11, 2021, which a) is related to, and claimsthe priority benefit of, U.S. Provisional Patent Application Ser. No.62/520,847, filed Jun. 16, 2017, b) is related to, and claims thepriority benefit of, U.S. Provisional Patent Application Ser. No.62/492,544, filed May 1, 2017, c) is related to, and claims the prioritybenefit of, U.S. Provisional Patent Application Ser. No. 62/488,042,filed Apr. 20, 2017, and d) is related to, claims the priority benefitof, and is a U.S. continuation-in-part patent application of, U.S.patent application Ser. No. 15/105,508, filed Jun. 16, 2016 and issuedas U.S. Pat. No. 10,314,686 on Jun. 11, 2019, which is related to, andclaims the priority benefit of, and is a U.S. § 371 national stagepatent application of, International Patent Application Serial No.PCT/US2014/070931, filed Dec. 17, 2014, which is related to, and claimsthe priority benefit of, a) U.S. Provisional Patent Application Ser. No.62/047,691, filed Sep. 9, 2014, and b) U.S. Provisional PatentApplication Ser. No. 61/917,051, filed Dec. 17, 2013. The entirecontents of the aforementioned applications are hereby incorporated byreference in their entirety into this disclosure.

BACKGROUND

Luminal or tubular grafts are useful for an extensive number of medicalapplications.

A. Small-Diameter Vascular Grafts

Vascular grafts, especially, are in high demand due to recent expansionsin the field. A major problem in vascular surgery is how to effectivelysupply blood to organs and tissues whose blood vessels are inadequateeither through congenital defects or acquired disorders such as trauma,arteriosclerosis or other diseases.

To date, the search for the ideal blood vessel substitute has focused onbiological tissues and synthetics. Initially, arterial homografts (humanarteries) were used to restore vascular continuity; however, limitedsupply, inadequate sizes, development of aneurysms and arteriosclerosisnecessitated the search for a better substitute. Additional substitutesthat have been employed include autologous blood vessels, vessels ofxenogenic origin, as well as vascular prostheses typically made fromDacron or polytetrafluoroethylene.

Despite intensive efforts to improve the nature of blood vesselsubstitutes, many problems with conventional substitutes remain. Forexample, conventional vascular grafts typically suffer from high failurerates related to (a) occlusion by thrombosis or kinking, or due to ananastomotic or intimal and subintimal hyperplasia (exuberant cell growthat the interface between the native vessel and graft); (b) a decreasingcaliber of the blood vessel substitute; (c) resulting infection; (d)biological failure or degradation; and/or (e) aneurysm formation. Otherproblems may involve compliance mismatches between the host vessel and asynthetic vascular prosthesis, which may result in anastomotic rupture,stimulated exuberant cell responses, and/or disturbed flow patterns andincreased stresses leading to graft failure.

Vascular grafts can be used in the treatment of numerous types ofmedical conditions, spanning a broad range of biological tissues. Forexample, and as described in further detail below, vascular grafts canbe employed in treating cardiovascular disease, obtaining vascularaccess for hemodialysis, as well as in nerve regeneration procedures.Unfortunately, conventional knowledge has yet to identify a functionalgraft that is capable of addressing the various biological issuesnecessary to maintain long term patency for these applications.

For example, cardiovascular disease, including coronary artery andperipheral vascular disease, is typically treated by surgicalreplacement. With around 8 million people with peripheral arterydisease, 500,000 patients diagnosed with end-stage renal disease, and250,000 patients undergoing coronary bypass surgeries each year in theUnited States alone, there is a significant demand for luminal grafts invascular surgery. This is especially true with respect to functionalsmall-caliber blood vessels (<4 mm in diameter).

Despite this clear clinical need for a functional small-diameter vesselgraft, replacement therapy with respect to small-diameter blood vesselshas been met with limited success. One reason or this is that theapplication of conventional methods for creating replacements forlarge-caliber vessels have generally proved inadequate when applied tosmall-caliber vessel substitutes. For example, while artificial,biological and modulated materials (including, without limitation,synthetic polymer scaffolds (polyurethane), synthetic scaffolds treatedwith biological molecules such as collagen, heparin, laminin,anti-coagulant peptides, etc.) have proven successful with respect tolarge-caliber vessel grafts, these materials are not particularly suitedfor creating small-diameter luminal grafts. This is due, at least inpart, to the lower blood flow velocities of smaller vessels, whichrequire a different set of design criteria and introduce a host of newproblems not encountered in large-caliber vessel substitutes. Indeed, inlow-flow situations, synthetic and other conventional grafts are proneto sudden thrombosis and provoking a wound-healing response fromadjacent vessels and the surrounding tissue that under somecircumstances narrows the lumen and reduces blood flow therethrough.Accordingly, when conventional materials are used to preparesmall-diameter vessel grafts, the replacement grafts' have shown anincreased tendency (a) for thrombogenicity; (b) to develop embolismand/or occlusion of the graft lumen (i.e. intimal hyperplasia andnegative remodeling); (c) to develop anastomotic intimal hyperplasia;(d) for aneurysm formation of the graft itself; and/or (e) to cause acompliance mismatch with the host vessel.

For these reasons, operations using autologous vessels remain thestandard for small-diameter grafts. However, there are also issuesassociated with this approach. Many patients do not have a vesselsuitable for use because of vascular disease, amputation, or previousharvest, and this method requires a second complicated surgicalprocedure to obtain the vessel. As a result, there is a demand for avascular prosthesis which is suited to the small-diameter blood vessels.

Recently, tissue engineering has emerged as an alternative approach toaddress the shortcomings of current options. Specifically,decellularized scaffolds (decellularized artery, vein and/or othersuitable tissue) have been made by removing the cellular components ofthe tissue, thereby resulting in a decellularized scaffold that isentirely comprised of natural extracellular matrix. After thedecellularized scaffold is formed, the same is recellularized by hostcells. For example, the scaffolds may host smooth muscle cells andfibroblasts that mimic native blood vessels. Purified proteins have alsobeen used to form scaffolds of such tubular constructs.

Preparation of the scaffolds typically requires a few months (aboutthree months) for the native smooth muscle and fibroblasts to seed onthe scaffold for inhibition of immunoreactions before implantation. Dueto the composition of such decellularized scaffolds, the scaffoldsretain beneficial native mechanical properties, promote regeneration andexhibit favorable biocompatibility. While over the last decade,cardiovascular tissue engineering has experienced a dramatic paradigmshift from biomaterial-focused approaches and towards the morebiology-driven strategies, there currently remains no functional vesselgraft that has addressed the various biological issues necessary tomaintain long term patency.

B. Hemodialysis

It has been estimated that, globally, approximately 8.3% of adults havediabetes and the number of people with diabetes is set to rise beyond592 million by 2025. Further, according to recent projections, 53.1million Americans will have diabetes in the year 2025 (diagnosed andundiagnosed), representing a 63% increase from the number of Americanswith diabetes today. As may be expected, the burden of cardiovasculardisease and premature mortality that is associated with diabetes willalso substantially increase, reflecting not only an increased amount ofindividuals with coronary artery disease, but an increased number ofyounger adults and adolescents with type 2 diabetes who are at a two- tofour-fold higher risk of experiencing a cardiovascular-related death ascompared to non-diabetics. Accordingly, aside from promoting awarenessand prevention of the disease, there is a vast need to facilitate bothtreatment and cost efficacy in the treatment of those afflicted with thechronic disorder.

Adults with diabetes or high blood pressure (or both) have an increasedrisk of developing chronic kidney disease (CKD). It has been estimatedthat more than 20 million Americans have CKD, including approximately 1of 3 adults with diabetes and 1 of 5 adults with high blood pressure.Other risk factors for CKD include cardiovascular disease, obesity, highcholesterol, lupus, and a family history of CKD. While some of thesepatients undergo treatment to maintain some kidney functions, somepatients lose their kidney function altogether, which is referred to asend-stage renal disease (ESRD). As the kidneys are responsible forfiltering out waste products from the blood, patients with ESRD requireeither dialysis or a kidney transplant to survive. Conventionally,three-times weekly, in-center dialysis is the most commonly performedmodality.

In 2012, it was estimated that around 398,000 Americans relied on someform of dialysis to keep them alive. Needless to say, the costassociated with providing such procedure is considerable. A significantportion of the total cost is spent on hemodialysis vascular access,which has been long considered to be the most problematic part ofdialysis. There are three basic kinds of vascular access forhemodialysis: 1) ateriovenous (AV) fistula; 2) an AV graft; or 3) avenous catheter. Hemodialysis patients who do not have adequate veinsfor a fistula become candidates for an AV graft or a venous catheter.Conventional AV grafts and venous catheters are typically discourageddue to their high morbidity and mortality. Specifically, such types ofvascular access tend to have more problems than fistulas with respect toclotting and infection.

An AV graft is created by connecting an artery to a vein with asynthetic tube of biocompatible material (i.e. the graft), andimplanting the same subcutaneously. The graft then functions as anartificial vein that can be used repeatedly for needle placement andblood access. One problem associated with this technique is thatthrombosis of the graft is common, which can develop due to poor bloodflow. Another risk relates to an increased risk in the development ofvascular access steal syndrome, which refers to vascular insufficiencyresulting from the AV graft. Considering the limitations of conventionalAV grafts and the prevalence of hemodialysis in the United States alone,a need exists for an improved design. Accordingly, it would be desirableto have a vessel graft that is capable of long term patency and does notincrease the risk of aneurysm.

C. Nerve Regeneration

Nerve injuries are common in clinical practice. Nervous system injuriesare most commonly caused by trauma, tissue loss, bone fracture, orburns. Severe nerve injury is either a segmental transection or defectresulting in partial or total loss of the motor, sensory, and autonomicfunctions conveyed by the injured nerves to the denervated segments ofthe body. In many cases, these injuries can lead to chronic functionaldisability as well as the development of neuropathic pain, both of whichcan have a devastating impact on patient quality of life.

While the central nervous system is, for the most part, incapable ofself-repair and regeneration, the peripheral nervous system (PNS) hasthe intrinsic ability to repair and regenerate. Specifically, nervefiber regeneration is due to the growth of transected axons of the nervestump proximal to the lesion and not to a regenerative process of axonsof the distal stump. Indeed, functional re-innervation requires that theaxons extend from the proximal nerve stump until reaching their distaltarget. However, PNS nerve regeneration is a complex biologicalphenomenon and typically does not occur spontaneously without treatment.Furthermore, nerves in the PNS can regenerate only under certaincircumstances. For example, while spontaneous natural regeneration mayoccur in peripheral nerve injuries if the distance to target is short,over large gaps, microsurgical repair is necessary.

Historically, the common surgical approach to repairing a transectednerve has been direct suture (or gluing) of the two stumps together whenthe ends can be approximated without tension. However, this technique isdifficult, time-consuming and often yields poor functional results.Furthermore, where nerve substance loss occurs (i.e. the defect islonger), a neurorrhaphy without tension at the site of repair cannot beperformed. For more extensive peripheral nerve injuries/lesions (e.g.,where the nerve defect gap is longer than, for example, about 20 mm) thesurgical repair of nerve gap has conventionally been achieved usingautologous nerve grafts harvested from other sites in the body. In suchcases, a nerve graft is typically used to bridge the two stumps or endsand promote nerve regeneration, rather than suturing the two stumpsunder tension. However, there are significant disadvantages to thisautologous nerve grafting technique as well as it requires an extraincision for the withdrawal of a healthy nerve (which could also resultin sensory residual deficits) and, often, the length of the graftmaterial is limited. Furthermore, sensory and motor neurons havedifferent Schwann cell modalities and, if placed in the incorrectmicroenvironment, may have limited regenerative ability. As such,autologous nerve grafting is limited to a critical nerve gap ofapproximately 5 cm in length. While allografts may be used for nervegaps of over 5 cm, allografts require the use of extensive immunesuppression up to 18 months post implantation.

Currently, biomedical strategies for PNS regeneration focus ondeveloping alternative treatments to nerve grafting (e.g., nerveguidance channels or tubulization), whereas efforts for spinal cordinjury are focused on creating a permissive environment forregeneration. Unfortunately, a solution to completely repair long spinalcord injuries has not yet been identified.

Sutureless tubulization techniques provide an alternative to directnerve sutures and nerve grafting. Tubulization involves formingnon-nervous luminal grafts (e.g., venous or arterial conduit grafts) tocreate optimal conditions for nerve regeneration over the empty spaceintentionally left between two nerve stumps. Nevertheless, suchalternatives have not shown substantial benefits compared with standardnerve grafts.

In order to achieve a better clinical outcome, various materials (bothbiological and synthetic) have been studied in connection withtubulization. Enriching the graft tubes with other tissue (e.g., piecesof nerve or skeletal muscle to form a “biological” graft) has seen somesuccess when used in tubulization applications, however, only withlimited efficacy—functional recovery has only been achieved for injuredgaps shorter than about 4 cm for both sensory and mixed nerves.Alternatively, non-biological synthetic materials have also beenemployed, albeit also with limited success. When nonabsorbable syntheticgrafts are used in humans, the occurrence of complications due to localfibrosis (triggered by the implant material) and nerve compressionbecomes a substantial concern. This is due, at least in part, to thegraft's non-degradable nature and its inability to adapt to the nervegrowth and maturation. As such, synthetic nerve repair conduits used forbridging strategies have increasingly been made of biodegradable orbioresorbable materials. Among these, polyglycolic acid nerve repairconduits are an example of one biodegradable material that has shown adecreased prevalence of complications as compared to non-absorbablesynthetic materials. However, even such bioabsorbable/bioresorbablenerve conduits have flaws and the results thus far are still notsatisfactory.

Finally, nerve reconstruction by tissue engineering has seen anincreased interest in recent years. In tissue engineering, two conceptshave guided the development of recent nerve regenerationtechnologies: 1) the manipulation of tissues and organs in vitro tofashion conduit should attempt to mimic important features of the nerveenvironment; and 2) various elements considered essential for promotingnerve fiber regeneration are missing in non-nerve grafts and, as such,an attempt should be made to enrich biological or synthetic tubes withthe same. However, currently, despite the ongoing research and workingconcepts, conventional conduits (whether formed by tissue engineering orotherwise) continue to fall short and exhibit critical flaws.Accordingly, it would be desirable to have a luminal graft thatsatisfies all of the biological requirements necessary for thesuccessful promotion of peripheral nerve regeneration.

In the Diabetes Mellitus and Chronic Kidney Disease patient, End StageRenal Disease (ESRD) is the terminal phase requiring kidney replacementtherapy for survival. Hemodialysis is the primary therapy for kidneyfunction replacement. In 2011, the United States Renal Disease System(USRDS) reports approximately 500,000 patients receiving hemodialysis.¹The USRDS reports expenditures for ESRD care approached S50 billion.

Vascular access of arterial blood is the preferred method forhemodialysis. For effective hemodialysis, maintaining a patent,high-flow vascular access site is the minimum requirement. In 2011,approximately $6.2 billion was spent just to keep existing vascularaccess sites open. For hemodialysis, the current solutions(arteriovenous fistula (AVF), arteriovenous graft (AVG), and tunneledcatheter (TC) do not offer overall satisfactory outcomes in theimmediate, short-term, or long-term. Patients must endure an accessmethod that introduces frequently occurring, significant and costlycomplications.

For the ESRD patient, hemodialysis is required to replace kidneyfunction, filtering waste and toxins from the blood, excreting excessfluids, and maintaining electrolyte balance. Hemodialysis requires asurgically created access site for the chronic application of the largebore needles, in which the blood is extracted, cleaned and balancedexternally, and then reinserted into the body. For the ESRD patient, itis essential to have a functioning vascular access site that cantolerate the multi-weekly dual-needle puncture injury while sustaininghigh blood flow.

Despite advances in long-term dialysis vascular access, a satisfactorysolution remains elusive for patients and physicians.

In recent years, the AVF has become the widely preferred first choicefor permanent vascular access because of a lower incidence of associatedmorbidity and mortality than other access types. Per the Kidney DiseaseOutcomes Quality Initiative (KDOQI) guidelines, the Rule of Three 6smust exist for vascular access, namely that the selected vein mustmature to allow 600 ml/min blood flow, growth to a diameter greater than6 mm, and resides less than 6 mm below the surface of the skin.

Fistulas require significant time to mature for surgical healing of theanastomosis site and wound, and more importantly vein adaptation(diameter enlargement and wall thickening) in response to the higharterial blood flow rate and pressure. Besides the lengthy time tomature, one of the primary drawbacks to AVFs is the high failure tomature rate (up to 57%) where the surgically created fistula neverenters service as an access site for dialysis.

The major advantage of AVF is for those that mature and functionallyprovide dialysis access; the subsequent complication and interventionrates are 7-fold lower than currently available AVGs. There is a widerange of failure rates in the literature ranging from 25% to 57%. In amulti-center prospective initiative by Huijbregts et al. of 491 newfistulas, the 1-year primary patency (from the day of surgical creationand includes failure to mature) was 49%. In a large meta-analysis(n=12,383), the 1-year primary patency (defined as creation of access tofirst AVF intervention to maintain or restore blood flow) and secondarypatency (defined as creation of access until access abandonmentincluding failures), was 60% and 71%. Primary failures were reported toat 23%. To date, the performance of AVFs in a level 1 randomizedclinical trial where an intent-to-treat approach to avoid treatment byindication bias has not been studied. The AVF solution may be thepreferred form of hemodialysis, but it is not without significantproblems.

Synthetic and autologous vascular grafts have been available forhemodialysis access for many years now. The expandedpolytetrafluoroethylene (ePTFE) with Heparin-bonded to the material hasbecome the preferred graft material choice. From a hemodialysis accesshierarchy standpoint, AVGs are a second or third choice behind a nativefistula or a second native fistula in another upper extremity location.Recent professional dialogue argues that recommendations originatingfrom KDOQI to prefer AVFs over grafts are largely based on single centerstudies patency results from the 1980's and early 90's that excludedthose fistulas that failed to mature. And in patients with compromisedforearm vessels, grafts have been shown to have higher patency thanAVFs. Grafts have distinct advantages over fistulas, namely anengineered diameter for appropriate blood volume, the time to becomeoperational is significantly shorter than fistula (1-2 weeks), lowfailure to mature rates, and lower tunneled catheter use. If thrombosisoccurs the clearing of clot is more predictable. Most importantly,functional secondary patency is equal to the fistula. Current AVG havesignificant disadvantages compared to AVFs, namely five times theinfection risk, lower primary patency, higher rates of thrombosis,higher intervention rates, a higher potential for pseudo-aneurysm(needle puncture sites due to material failure), stenosis at the venousanastomosis, and a 2.5× higher mortality rate (this position ischallenged in the literature as it hypothesized that sicker patientsreceive more grafts). Despite their shortcomings of thrombosis andinfection, the advantages of dialyzing quickly with low failure ratesand comparable secondary patency rates positions AVG as a viable option.

TC are the temporary option for hemodialysis during which an AVF or AVGis given time to mature. During this lag time for the permanent accesssolutions to become operational, a dual-lumen catheter, usually placedin the non-dominant internal jugular and terminating in the superiorvena cava, is inserted to enable dialysis in the short-term. They mayalso be used as permanent access when a patient lacks a suitable sitefor access. The advantage for TC is that dialysis can begin immediately,but there exists several significant shortcomings, including primarydysfunction, frequent low flow due to thrombosis, fibrin collecting onthe catheter tip or an anatomically malposition tip, central veinstenosis/thrombosis, or infection originating at the external site. Eachshortcoming requires intervention ranging from a simple catheter-lumenthrombolysis to a radiologic procedure and/or extensive antibiotictherapy. Infection of the tunneled catheter is a common complication.When the catheter is left in place less than 2 weeks, incidence ofinfection is less than 5%. However, incidence increases to 25% withlonger placement. The clinical need for a solution that can be readilycreated, mature at a high rate, begin dialysis quickly, demonstrate lowearly and late thrombosis rates, exhibit low neointimal hyperplastictendencies, and yield long-term access is clearly evident.

BRIEF SUMMARY

In at least one exemplary embodiment of a luminal graft (also referredto as a blood vessel graft in various embodiments herein that pertain toblood vessel grafting in particular) of the present disclosure, thegraft comprises a generally tubular element configured for plasma and/orblood cells to flow therethrough. The tubular element is comprised ofelastin and collagen fibers, with the elastin fibers being a dominantcomponent thereof. Alternatively, or additionally, the tubular elementmay comprise a ratio of collagen fibers to elastin fibers ranging fromabout a 1.2 ratio value to about a 0.8 ratio value. For example, in atleast one embodiment, the collagen to elastin ratio may comprise about a1:1 ratio. Alternatively, the collagen to elastin ratio may compriseabout a 1.10 ratio value. In at least one exemplary embodiment, thebiological tissue comprised pulmonary ligament tissue or visceral pleuratissue. Additionally or alternatively, the at least one layer maycomprise a biological tissue comprising between about 11% and about 12%collagen fibers and between about 12.5% and 13.5% elastin fibers. In yetanother embodiment, the biological tissue exhibits mechanical propertiesthat are similar pre- and post-fixation. Additionally or alternatively,in at least one embodiment, the generally tubular element is flexible.

Where the luminal graft comprises at least one layer, each of the layersmay comprise a first edge and a second edge, both of which extendbetween the proximal and distal ends of the layer. Additionally, each ofthe layers may comprise a seam extending between the proximal and distalends thereof, the seam comprising the first and second edges sealedtogether via one or more closure mechanisms. For example, the closuremechanism(s) may comprise an arrow-lock configuration, magnetic strips,a series of perforations and sutures, and/or a series of clips.Additionally or alternatively, the closure mechanism may comprisesutures.

In at least one embodiment, the at least one layer of the generallytubular element comprises three concentric layers. There, at least oneof the layers may be comprised of a synthetic material and at least oneof the layers is comprised of pulmonary ligament or pulmonary visceralpleura. In those embodiments having more than one layer, the seam ofeach of the layers may be offset from the seam of each of theimmediately adjacent layer(s). While such offset may comprise any angle,in at least some embodiments, the offset comprises between about aninety degree angle and about a one hundred and eighty degree angle.

In yet another embodiment, the lumen of the generally tubular elementcomprises at least one diameter that is equal to or less than about 5 mmor equal to or less than about 1 mm.

Still further, the generally tubular element may comprise a luminalsurface having mesothelium thereon. Additionally, the generally tubularelement may be configured to allow cells from an adjacent blood vesselto integrate within the fibers thereof and thus remodel the same whenthe graft is implanted within a mammalian body.

Additional embodiments of the luminal graft of the present disclosureare formed by wrapping at least one layer around a mandrel having acylindrical configuration and at least one diameter to form thegenerally tubular element; coupling at least one closure mechanism withthe at least one layer to form a seal along the length of the layer; andwithdrawing the mandrel from the generally tubular element. The at leastone diameter of the tubular element may be substantially equivalent tothe at least one diameter of the mandrel. In at least one embodiment,the at least one diameter of the mandrel comprises about or less than 5mm, or even about or less than 1 mm. The seam of the at least one layermay comprise a minimal profile. Additionally or alternatively, the graftmay be further formed by wrapping at least one additional layer aroundthe mandrel and the previously wrapped layer and coupling at least oneclosure mechanism with each additional layer to form a seam along thelength thereof. For example, in at least one embodiment, the generallytubular element comprises three concentric layers. Still further, atleast one of the layers may comprise pulmonary ligament tissue orpulmonary visceral pleura tissue and, optionally, one of the additionallayers may comprise a tissue or material other than pulmonary visceralpleura. For example, at least the inner-most layer may comprise visceralpleura and, in at least one embodiment, a luminal surface of theinner-most layer comprises mesothelium.

In at least one exemplary embodiment of a luminal graft of the presentdisclosure, the graft comprises at least one layer formed into agenerally tubular element that may or may not be flexible. The tubularelement has a proximal end, a distal end and a lumen extendingtherebetween, and is configured so that passage of plasma and bloodcells into or through the lumen is permitted. Furthermore, in at leastone embodiment the layer comprises a biological tissue comprisingelastin and collagen fibers, where the elastin fibers are the dominantcomponent thereof. Additionally or alternatively, the biological tissuemay comprise between about 11% and about 12% collagen fibers and betweenabout 12.5% and 13.5% elastin fibers. Still further, the biologicaltissue may exhibit mechanical properties that are similar pre- andpost-fixation. In at least one exemplary embodiment, at least one of thelayers comprises a biological tissue such as pulmonary ligament tissueand/or visceral pleura. Accordingly, the tubular element of the luminalgraft may comprise in inner wall (i.e. a luminal surface) havingmesothelium thereon. Additionally, in another exemplary embodiment, thelumen of the tubular element comprises a diameter that is equal to orless than about 5 mm. In addition to a layer of biological tissue, atleast one of the additional layer(s) of the graft may comprise asynthetic material.

In another embodiment, each layer of the luminal graft comprises a firstedge and a second edge. Both the first and second edges extend betweenthe proximal and distal ends of the layer. Furthermore, the luminalgraft further comprises a seam extending between the proximal and distalends of the layer, the seam comprising the first and second edges sealedtogether via one or more closure mechanisms. The closure mechanism(s)may comprise an arrow-lock configuration, magnetic strips, a series ofperforations and sutures, and/or a series of clips.

In at least one embodiment, the at least one layer of the graftcomprises three concentric layers. There, the seam of each of the threeconcentric layers may be offset from the seam of each of the immediatelyadjacent layer(s). Further, in at least one embodiment, the offsetcomprises about a ninety degree angle.

In at least one embodiment of a system for the manufacture of a luminalgraft, the system comprises a mandrel having a cylindrical configurationand at least one diameter, at least one layer comprising a length, andat least one closure mechanism configured to couple with the layer toform a seam along the length of the layer. Here, when the layer ispositioned around the mandrel, a generally tubular element having atleast one diameter that is substantially equivalent to the at least onediameter of the mandrel is formed. For example, and without limitation,the first diameter of the mandrel may comprise less than or equal toabout 5 mm. Furthermore, at least one layer of the system may compriseat least three layers and, in at least one exemplary embodiment, atleast one of the layers comprises pulmonary ligament tissue and/orvisceral pleura oriented such that mesothelium faces the lumen of thetubular element.

In at least one exemplary embodiment of a method for manufacturing aluminal graft of the present disclosure, the method comprises the stepsof (a) wrapping a first layer around a mandrel having at least onediameter; (b) positioning a closure mechanism coupled with the firstlayer for deployment; (c) engaging and/or deploying the closuremechanism, thereby forming a seam along a length of the first layer anddefining a generally tubular element having at least one diameter thatis substantially equal to the at least one diameter of the mandrel; (d)minimizing the profile of the seam; and (e) withdrawing the mandrel fromthe first layer. Where the luminal graft comprises more than one layer,the method for manufacturing the same may further comprise repeatingsteps (a)-(d) for the additional layers as necessary. Furthermore, themethod may further comprise the step of ensuring a surface of the firstlayer comprising mesothelium is positioned facing the mandrel.

Methods for performing a luminal grafting procedure are also disclosed.In at least one embodiment, the method comprises the steps of:implanting a luminal graft within a mammalian body at a location of anarterial anastomosis, the luminal graft comprising at least one layerformed into a generally tubular element having a proximal end, a distalend and a lumen extending therebetween and configured such that passageof plasma and/or blood cells into or through the lumen is permitted, andwherein at least one of the layers comprises biological tissuecomprising elastin and collagen fibers, with elastin being a dominantcomponent thereof; providing at least an initial barrier betweenendothelial and smooth muscle cells of the artery using the luminalgraft; and facilitating a remodeling process such that the smooth musclecells of the artery integrate into the luminal graft. For example, theluminal graft may be configured to remodel pursuant to a physiologicalremodeling process of the mammalian body.

The biological tissue of the luminal graft used in the method of thepresent disclosure may comprise pulmonary ligament tissue and/orvisceral pleura. Further, each of the layers of the luminal graft maycomprise a first edge and a second edge, both of which extend betweenthe proximal and distal ends of the layer. Additionally, a seam mayextend between the proximal and distal ends of the layer, such seamcomprising the first and second edges sealed together via one or moreclosure mechanisms.

In at least one alternative embodiment, the anastomosed artery comprisesa small-diameter vessel and the lumen of the luminal graft comprises atleast one diameter that is equal to or less than about 5 mm. In yetanother embodiment, the lumen of the luminal graft comprises at leastone diameter that is equal to or less than about 1 mm.

In additional embodiments of the method for performing a luminalgrafting procedure, at least one of the layers of the luminal graftcomprises a synthetic material, the tubular element of the luminal graftmay further comprises a luminal surface having mesothelium thereon, atleast one closure mechanism of the luminal graft may further compriseone or more sutures and/or the at least one closure mechanism of theluminal graft may comprise an arrow-lock configuration, magnetic strips,a series of perforations and sutures, and/or a series of clips.

In at least one exemplary embodiment of a luminal graft of the presentdisclosure, the luminal graft comprises at least an inner layer formedinto a generally tubular element having a proximal end, a distal end,and a lumen extending therebetween. The inner layer of the luminal graftcomprises biological tissue and additionally has a luminal surfacehaving mesothelium thereon. In at least one alternative embodiment, theluminal graft additionally comprises at least one additional layerpositioned concentrically around the inner layer.

In at least one embodiment, the biological tissue comprises elastin andcollagen fibers, with elastin being a dominant component. In at leastone embodiment, the biological tissue used for the luminal graft maycomprise pulmonary pleura, parietal pleura, pleura ligament tissue, andmediastinal pleura, or a combination thereof.

In yet another embodiment, the lumen of the tubular element comprises atleast one diameter that is equal to or less than about 4 mm (i.e. asmall-diameter graft). The tubular element of the graft may be flexibleand, in at least one embodiment, the luminal surface of the inner layeris capable of attenuating the growth of scar tissue and exhibitsanti-thrombotic and anti-adhesive properties. Additionally, the tubularelement of the luminal graft is capable of arterialization.

In certain embodiments, the luminal graft may comprise a cell guidanceconduit and each of the proximal and distal ends of the tubular elementmay be configured to receive a nerve stump. Alternatively, in otherembodiments, the luminal graft may comprise an arteriovenous graft.There, the proximal end of the tubular element may be configured toreceive blood flow into the lumen from an artery, the distal end of thetubular element may be configured for placement into a vein, and thelumen of the tubular element may be configured for passage of blood fromthe proximal end to the distal end.

In at least one exemplary embodiment of a method for promotingregeneration of a damaged nerve, the method comprises the steps of: (a)providing a luminal graft comprising an inner layer formed into agenerally tubular element having a proximal end, a distal end, and alumen extending therebetween, wherein the inner layer comprisesbiological tissue and a luminal surface having mesothelium thereon; and(b) placing the luminal graft at a site of neuronal injury to facilitateregeneration of the nerve. In certain embodiments of the aforementionedmethod, the biological tissue of the inner layer comprises elastin andcollagen fibers, with elastin being a dominant component thereof.Furthermore, the biological tissue may be selected from a groupconsisting of pulmonary pleura, parietal pleura, pleura ligament tissue,and mediastinal pleura, or comprise any combination thereof (where, forexample, the luminal graft comprises multiple layers). In certainembodiments, the method of the present application may further comprisethe step of maintaining patency of the tubular element for at least 6months such that the nerve is allowed to regenerate within the lumenthereof.

In additional embodiments of the method, the lumen of the tubularelement comprises at least one diameter that is equal to or less thanabout 4 mm. Additionally or alternatively, the tubular element may beflexible and the luminal surface of the inner layer may be capable ofattenuating the growth of scar tissue and exhibits anti-thrombotic andanti-adhesive properties. In yet another exemplary embodiment, the nerveof the method is a mammalian peripheral nerve. Furthermore, in certaincases where the nerve is severed and comprises a first severed end and asecond severed end, the method may further comprise the steps of: (c)bringing the first severed end of the nerve into contact with theproximal end of the tubular element; and (d) bringing the second severedend of the nerve into contact with the distal end of the tubular elementso as to bridge the neuronal injury such that the lumen of the tubularelement is substantially collinear with the first and second severedends of the nerve. Additionally, in at least one embodiment stemmingfrom the aforementioned, the ends of the severed nerve are sutured toeach respective end of the tubular element.

In yet another exemplary embodiment of the present disclosure, a methodfor providing vascular access in connection with the delivery ofhemodialysis to a patient is provided. In at least one embodiment, themethod for providing vascular access in connection with the delivery ofhemodialysis to a patient comprises the steps of: (a) providing aluminal graft comprising an inner layer formed into a generally tubularelement having a proximal end, a distal end, and a lumen extendingtherebetween, and wherein the inner layer comprises biological tissueand a luminal surface having mesothelium thereon and at least the innerlayer is capable of arterialization; (b) implanting the luminal graft ina patient to achieve vascular access such that the luminal graftconnects an artery of the patient to a vein of the patient; (c)establishing blood flow from the artery to the vein through the lumen ofthe tubular element; and (d) allowing at least the inner layer toarterialize over time while being subjected to continuous blood flowthrough the lumen of the tubular element. Additionally, the method maycomprise the additional steps of: (i) inserting the distal end of thetubular element of the luminal graft through an incision into the veinsuch that the distal end of the tubular element passes to a pointdownstream of the incision; (ii) surgically securing the luminal graftto the vein; (iii) anastomosing the proximal end of the tubular elementto a preselected artery such that blood flow is established through thelumen of the tubular element, the blood flow entering the lumen throughthe proximal end of the tubular element and exiting the lumen throughthe distal end of the tubular element; and (iv) allowing at least theinner layer to arterialize over time. Moreover, in at least oneembodiment, the method may further comprise the step of maintainingpatency of the tubular element for at least 6 months while subjectingthe luminal graft to continuous blood flow therethrough.

The present disclosure includes disclosure of a luminal graft comprisinga generally tubular element configured for nerve cells to growtherethrough, the luminal graft comprising:

at least one sheet of biological tissue having elastin fibers andcollagen fibers, with the elastin fibers being a dominant componentthereof; and

a plurality of microchannels formed on a surface of the at least onesheet of biological tissue, each of the microchannels extendinglongitudinally between a first end and a second end of the at least onesheet of biological tissue and configured to provide intraluminalstructural guidance to nerve cells proliferating therethrough.

The present disclosure includes disclosure of a luminal graft, whereinthe biological tissue comprises pulmonary ligament tissue or visceralpleura tissue.

The present disclosure includes disclosure of a luminal graft, furthercomprising a plurality of biodegradable microspheres affixed to thesurface of the at least one sheet of biological tissue and positioned inrows extending longitudinally between the first end and the second endof the at least one sheet of biological tissue, wherein the rows ofmicrospheres define side portions of the plurality of microchannels.

The present disclosure includes disclosure of a luminal graft, whereineach of the biodegradable microspheres encapsulates a substance and,when the biodegradable microsphere degrades, the substance is releasedtherefrom.

The present disclosure includes disclosure of a luminal graft, whereinthe substance comprises a neurotrophic factor.

The present disclosure includes disclosure of a luminal graft, whereinthe plurality of biodegradable microspheres further comprises a firstset of biodegradable microspheres encapsulating a first neurotrophicfactor and a second set of biodegradable microspheres encapsulating asecond neurotrophic factor.

The present disclosure includes disclosure of a luminal graft, whereinthe first neurotrophic factor comprises NGF and the second neurotrophicfactor comprises NT-3.

The present disclosure includes disclosure of a luminal graft, whereinthe plurality of biodegradable microspheres further comprises a firstset of biodegradable microspheres that have a first rate of degradationand a second set of biodegradable microspheres that have a second rateof degradation; wherein the first rate of degradation is faster than thesecond rate of degradation.

The present disclosure includes disclosure of a luminal graft, whereinthe first set of biodegradable microspheres is positioned at or near thefirst and second ends of the at least one sheet of biological tissue andthe second set of biodegradable microspheres is positioned in a middleportion of the at least one sheet of biological tissue.

The present disclosure includes disclosure of a luminal graft, whereinthe at least one sheet of biological tissue is arranged to form cylindercomprising a plurality of internal layers, each layer comprising aplurality of biodegradable microspheres positioned in rows extendinglongitudinally between the first end and the second end of the sheet,wherein the rows of microspheres define side portions of the pluralityof microchannels.

The present disclosure includes disclosure of a grating module forfabricating a luminal graft comprising a plurality of microspheres, thegrating module comprising a series of notches, each notch separated byan interval and comprising a depth and a width, wherein the depth isabout 20% smaller than the width.

The present disclosure includes disclosure of a grating module, whereineach notch comprises a length of 6 cm, the width of each notch isselected from the group consisting of 100 μm, 200 μm, and 400 μm, andthe depth of each notch is selected from the group consisting of 80 μm,160 μm, and 320 μm, respectively.

The present disclosure includes disclosure of a method for performing aluminal grafting procedure, the method comprising the steps ofimplanting a luminal graft within a mammalian body at a location of aproximal nerve stump and a distal nerve stump, the luminal graftcomprising at least one sheet of biological tissue having elastin fibersand collagen fibers, with the elastin fibers being a dominant componentthereof, a plurality of microchannels formed on a surface of the atleast one sheet of biological tissue, each of the microchannelsextending longitudinally between a first end and a second end of the atleast one sheet of biological tissue and configured to provideintraluminal structural guidance to nerve cells proliferatingtherethrough, and a plurality of biodegradable microspheres affixed tothe surface of the at least one sheet of biological tissue andpositioned in rows extending longitudinally between the first end andthe second end of the at least one sheet of biological tissue, whereinthe rows of microspheres define side portions of the plurality ofmicrochannels and each of the microspheres encapsulates a neurotrophicfactor, wherein the at least one sheet of biological tissue is arrangedinto a cylindrical configuration such that the plurality ofmicrochannels comprise a ceiling and a floor formed from the at leastone sheet of biological tissue.

The present disclosure includes disclosure of a method, furthercomprising the steps of releasing a first set of neurotrophic factorsfrom a first set of the microspheres to facilitate nerve proliferationat or near the first end and the second end of the at least one sheet ofbiological tissue; and releasing a second set of neurotrophic factorsfrom a second set of the microspheres to facilitate nerve proliferationin a middle portion of the at least one sheet of biological tissue.

The present disclosure includes disclosure of a method, wherein the stepof releasing a first set of neurotrophic factors occurs at a first timeand the step of releasing a second set of neurotrophic factors occurs ata second time, the second time occurring after the first time.

The present disclosure includes disclosure of a hybrid luminal graftcomprising a generally tubular element, the luminal graft comprising afirst layer comprising one or more synthetic materials, the first layerdefining an inner surface and an opposing outer surface; and abiological material applied to the inner surface of the first layer;wherein when the first layer is configured as a generally tubularelement, the biological material is present within a defined lumen ofthe first layer.

The present disclosure includes disclosure of a hybrid luminal graft,wherein the synthetic material is selected from the group consisting ofsilicone, polytetrafluoroethylene, and elastomer.

The present disclosure includes disclosure of a hybrid luminal graft,wherein the biological material is selected from the group consisting ofpulmonary visceral pleura, pulmonary ligament, a component harvestedfrom pulmonary visceral pleura, and a component harvested from pulmonaryligament.

The present disclosure includes disclosure of a hybrid luminal graft,wherein a biological glue is used to facilitate adherence of thebiological material to the inner surface.

The present disclosure includes disclosure of a hybrid luminal graft,wherein the biological material comprises glycocalyx.

The present disclosure includes disclosure of a hybrid luminal graft,further comprising a second layer comprising one or more syntheticmaterials, the second layer positioned around a relative outside of thefirst layer.

The present disclosure includes disclosure of a hybrid luminal graft,further comprising a third layer comprising one or more syntheticmaterials, the third layer positioned around a relative outside of thesecond layer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of a portion of an exemplary embodimentof a luminal graft suitable for the replacement of small-diametervessels according to the present disclosure;

FIG. 2A shows a perspective, exploded view of a portion of an embodimentof a luminal graft having three layers;

FIG. 2B shows a perspective view of an embodiment of a luminal graft ofthe present disclosure comprising a rolled configuration formed from asingle sheet of tissue;

FIGS. 3A and 3B show an embodiment of an arrow-locking closure mechanismof a luminal graft according to the present disclosure;

FIGS. 4A and 4B show an embodiment of a magnetic closure mechanism of aluminal graft according to the present disclosure;

FIGS. 5A and 5B show an embodiment of a perforated closure mechanism ofa luminal graft according to the present disclosure;

FIG. 6A shows an embodiment of a clamp closure mechanism of a luminalgraft according to the present disclosure;

FIG. 6B shows a cross-sectional view of the clamp closure mechanism ofFIG. 6A;

FIG. 7 shows a perspective view of the luminal graft of FIG. 1;

FIGS. 8A-8D show embodiments of a system for manufacturing a luminalgraft according to the present disclosure;

FIG. 9 shows a flow chart depicting various steps of a method formanufacturing a luminal graft according the present disclosure;

FIG. 10A shows two-photon microscopy and fluorescent images of theultrastructure of a pulmonary visceral pleura tissue sample, withelastin fiber (left), collagen fibers (middle), and merged images(right);

FIG. 10B shows a graphical representation of the elastin fiber (E, n=10)and collagen fiber (C, n=10) content of pulmonary visceral pleuratissue;

FIG. 10C shows representative images of fibroblast attachment topulmonary visceral pleura tissue (PVP, left) and small intestinesubmucosa (SIS, right), and highlights reduced fibroblast adhesion topulmonary visceral pleura as compared to small intestine submucosa;

FIG. 10D shows representative images of pulmonary visceral pleura (PVP)and small intestine submucosa (SIS) that illustrates reducedcytotoxicity of PVP as compared to SIS;

FIG. 11A shows a schematic of in vivo remodeling of a pulmonary visceralpleura graft (E—endothelial cells, SMC—smooth muscle cells, IEL—internalelastic lamina, ADV—adventitia);

FIG. 11B displays immunofluorescent images of a pulmonary visceralpleura graft cross-section at 10 days and 12 weeks in vivo (20×objective; Red-Elastin, Blue-Nuclei);

FIG. 12A shows a side view representing a 0.80 mm pulmonary visceralpleura (PVP) graft prior to implantation;

FIG. 12B shows a graphical representation of the functional response ofthe PVP graft of FIG. 12A to pharmacological vasodilation andconstriction after 6 months of in vivo remodeling (Acetylcholine, ACh;Sodium Nitroprusside, SNP; Endothelin-1, ET1; concentrations in mol/L).

FIG. 13A shows a transmission electron microscope image of across-section of pulmonary pleura;

FIG. 13B shows a scanning electron microscope image of the pulmonarypleura shown in FIG. 3A;

FIGS. 14A and 14B show A) a luminal graft according to the presentdisclosure being used as a nerve guidance conduit, and B) the end of thenerve guidance conduit of FIG. 14B;

FIG. 14C shows a close-up view of a sample of pulmonary pleura in vivo;

FIGS. 15A, 15B, and 15C show histological images of a graft implanted ina femoral artery for 24 weeks, with FIG. 15A showing an optical image,FIG. 15B showing a transmission electronic microscopy image, and FIG.15C showing a scanning electron microscopy image, according to thepresent disclosure;

FIGS. 16A and 16B show traverse cross-sectional views of a luminal graftaccording to the present disclosure comprising microchannels;

FIG. 16C shows a schematic of a microchannel of the luminal graft ofFIG. 16B;

FIG. 17 shows a perspective view of a biological sheet of the presentdisclosure coated with microspheres to form microchannels within aluminal graft according to the present disclosure;

FIG. 18 shows a perspective view of a grating module for fabricating theluminal graft comprising microspheres according to the presentdisclosure;

FIG. 19 shows a perspective view of the grating module of FIG. 18 beingused to coat the biological sheet of FIG. 17 with microspheres to formmicrochannels;

FIG. 20 shows a perspective view of a sheet coated with microspheresaccording to the present disclosure rolled up to form a cylindricalluminal graft;

FIGS. 21A and 21B show perspective views of the steps of a method forfabricating a multi-layered cylindrical luminal graft according to thepresent disclosure.

FIG. 22 shows a perspective, exploded view of a portion of an embodimentof a luminal graft having three layers;

FIGS. 23 and 24 show side views of elements of grafts prior to beingconfigured as tubular elements, according to the present disclosure;

FIGS. 25 and 26 show grafts in tubular or cylindrical form, according toembodiments of the present disclosure; and

FIG. 27 shows a block component diagram of elements of a mixture of thepresent disclosure.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended. On the contrary,this disclosure is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of thisapplication as defined by the appended claims. As previously noted,while this technology may be illustrated and described in one or morepreferred embodiments, the devices, systems and methods hereof maycomprise many different configurations, forms, materials, andaccessories.

For example, the novel systems, methods and techniques of the presentapplication will be described in the context of replacing damaged orcompromised blood vessels and engineering luminal grafts for variousmedical applications. Unlike conventional luminal grafts, the inventivegrafts of this disclosure are capable of not only maintaining long termpatency, but also arterializing over time. While the luminal graftsdescribed herein may be prepared having any diameter, it will beappreciated that the inventive blood vessel grafts of this disclosuremay be configured and functional for the replacement of small-diameterblood vessels. Indeed, the blood vessel grafts described herein do notexperience the problems associated with small-diameter constructsprepared in accordance with conventional methods such as stenosis,aneurysm, or thrombosis formation. Furthermore, while the systems andmethods described herein are suitable for preparing blood vessel grafts(exemplary luminal grafts of the present disclosure as useful inconnection with blood vessels), having any diameter, such systems andmethods are especially suited for preparing blood vessel grafts havingat least a portion with a small diameter (e.g., less than or equal toabout 5 mm). Additionally, it is contemplated that the inventiveconcepts underlying the grafts, systems and methods described herein mayalso be applied to other tissue engineering applications such as, andwithout limitation, venous valves, microvessels, nerve grafts, duramatter, and stent coverings. Indeed, certain embodiments of the presentdisclosure provide a novel nerve guidance conduit that is abundant inextracellular matrix (elastin, collagen, and glycocalyx) for providingchemical cues for regenerating axons, provides intraluminalmicrochannels for topographic guidance cues for regenerating axons, andallows for the spatial and/or temporal release of neurotrophic factorsto further promote axonal regeneration. Importantly, such guidanceconduits show significant promise in not only promoting axonalregeneration for shorter nerve injury gaps, but also in larger gaps aswell.

Because of these unique and advantageous properties, and as will bedescribed herein in further detail, the luminal grafts of the presentdisclosure are particularly well suited for use in, and are functionalfor, vascular replacement therapies (small-diameter and otherwise),anastomosis formation, tubulization or nerve regeneration therapies, andarteriovenous graft hemodialysis. Additionally, while the luminal graftsdescribed herein may be prepared having any diameter, it will beappreciated that the inventive grafts of this disclosure may beconfigured and are functional for the replacement of small-diameterblood vessels (e.g., vasculature having at least a portion with adiameter of less than or equal to about 4 mm) or for use assmall-diameter conduits (e.g., conduits having at least a portion with adiameter of less than or equal to about 4 mm). Likewise, the systems andmethods described herein are suitable for preparing grafts and conduitshaving any diameter, including but not limited to those grafts andconduits having at least a portion with a small diameter (e.g., lessthan or equal to about 4 mm).

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present disclosure.Particular examples may be implemented without some or all of thesespecific details. In other instances, well known harvesting, processing,and storing operations have not been described in detail so as to notunnecessarily obscure the present disclosure.

Various techniques and mechanisms of the present disclosure willsometimes describe a connection between two components. Words such asattached, affixed, coupled, connected, and similar terms with theirinflectional morphemes are used interchangeably, unless the differenceis noted or made otherwise clear from the context. These words andexpressions do not necessarily signify direct connections, but includeconnections through mediate components and devices. It should be notedthat a connection between two components does not necessarily mean adirect, unimpeded connection, as a variety of other components mayreside between the two components of note. For example, a closuremechanism may be connected to a graft, but it will be appreciated thatone or more components may reside between the actual graft layer and theclosure mechanism. Consequently, a connection does not necessarily meana direct, unimpeded connection unless otherwise noted.

Furthermore, wherever feasible and convenient, like reference numeralsare used in the figures and the description to refer to the same or likeparts or steps. The drawings are in a simplified form and not to precisescale. For example, the disclosure and Figures of the presentapplication for the most part reference the inventive luminal graftsdescribed herein as having a single diameter or as being “small-diameterblood vessels.” It is understood that the disclosure is presented inthis manner merely for explanatory purposes and the principles andembodiments described herein may be applied to grafts that have varyingdiameters along their lengths (as well as the systems and methods formanufacturing the same). For example, the disclosure hereof may beapplied to luminal grafts that are longer than about 30 mm in length andconfigured to have a varying diameter so as to more accurately mimic anative blood vessel (i.e. the distal diameter may be smaller than theproximal diameter). In fact, as described herein, the use of pulmonaryligament tissue, visceral pleura, and/or mediastinal pleura in thecomposition of a varying diameter luminal graft can have a particularlyadvantageous effect, especially where the smallest diameter thereof isless than or equal to about four millimeters (≤4 mm) or is less than orequal to about five millimeters (≤5 mm)

FIG. 1 shows a perspective view of at least one embodiment of anexemplary luminal graft 10. Luminal graft 10 comprises a tubularconstruct having a diameter D and one or more concentric layers 12.Diameter D (also considered to be a perimeter in the event luminal graft10 is not absolutely circular in cross-section) of the graft 10 maycomprise any diameter of a vessel in need of replacement or of a desiredconduit, but in at least one exemplary embodiment, diameter D is lessthan or equal to about four or five millimeters (≤4 or ≤5 mm). Forexample, in at least one exemplary embodiment, diameter D of the graft10 may be/comprise three millimeters or less than about threemillimeters (≤3 mm) or even may be/comprise eight tenths of a millimeteror less than about eight tenths of a millimeter (≤0.8 mm).

Each of the one or more layers 12 of the graft 10 comprises a first edge16 and a second edge 18, as shown in FIG. 1, and an inner surface 17 andan outer surface 19, as shown in FIG. 2. The layer 12 is shaped todefine a lumen 14 extending the length thereof, where the first andsecond edges 16, 18 are positioned proximally to each other, the innersurface 17 faces the lumen 14, and the outer surface 19 faces outwardly.The first edge 16 and the second edge 18 are securely sealed togethervia one or more closure mechanisms 22 to form a seam 20 extending thelength of the luminal graft 10.

In the exemplary embodiment shown in FIG. 1, luminal graft 10 comprisesone layer 12; however, luminal graft 10 may comprise any number ofconcentric layers 12 wrapped around each other. For example, and withoutlimitation, FIG. 2 illustrates at least one embodiment of the luminalgraft 10 having three layers 12 (it will be understood that FIG. 2illustrates an exploded view of the luminal graft 10 to clearlyillustrate the various layers 12; in actuality, such layers 12 are incontact with each other and wrapped tightly together). The number oflayers 12 and/or dimensions of the luminal graft 10 may be selecteddepending upon the desired wall thickness of the luminal graft 10 orpursuant to a particular application or patient specifications. Forexample, where a graft 10 having a thicker wall is desired (such as forreplacement of a portion of a larger vessel), the luminal graft 10 maycomprise multiple layers 12. Alternatively, where a thinner wall is moreappropriate, the graft 10 may comprise fewer layers 12. The thickness ofeach individual layer 12 may also be selected to achieve a desiredoverall wall thickness of the graft 10 and/or to affect the propertiesthereof (by way of a non-limiting example, the inner-most layer 12 maybe thicker than the outer-most layer 12). In this manner, inapplication, the overall wall thickness of the luminal graft 10 can bematched to the native blood vessel or nerve of interest, for example,such that the ratio of wall thickness to diameter is nearly constant(e.g., thickness may be about ten percent (10%) of graft 10 diameter atany given point and assuming about a +/−20% acceptable variance rangebetween the graft and native blood vessel). In the case of nerveconduit, thickness may be at or about thirty percent (30%) of graft 10diameter and the diameter of nerve conduit is at or about three to eightpercent (3-8%) larger than native nerve to allow insertion if nativenerve stump/end into the lumen of the nerve conduit.

Furthermore, where a luminal graft 10 comprises more than one layer 12,the layers 12 may be positioned relative to each other such that theseams 20 thereof are positioned in an offset configuration. For exampleand without limitation, in the embodiment shown in FIG. 2A, the seams 20of the layers 12 are alternated at roughly one hundred and eighty degree(180°) angles to ensure an “air tight”—i.e. leak proof—construct.Additionally or alternatively, the seams 20 may be offset at otherdegrees as well, including ninety degrees (90°) or any degree of offset,or combinations thereof, that may be desired.

Other embodiments of the present disclosure contemplate a graft 10comprising a single sheet of tissue that is simply rolled up to providea multi-layered cylinder (see FIG. 2B). This rolled configurationminimizes the number of seams 20 within the luminal graft 10, whilestill providing a multi-layered structure.

In at least one exemplary embodiment of the present disclosure, the oneor more layers 12 of luminal graft 10 are comprised of a thin scaffoldof biological tissue that consists largely of elastin and some collagenfibers (the converse of small intestine submucosa (SIS)) (Conventionalmethodologies have previously evaluated fixed acellular biomaterialssuch as the pericardium and SIS as small-diameter vessel grafts. Whileboth of these biomaterials showed promise as large diameter vesselgrafts, neither remained patent as small-diameter vessel grafts and, assuch, are not conventionally used in coronary artery bypass graftingsurgery). For example, and without limitation, the one or more layers 12of luminal graft 10 may comprise pulmonary ligament tissue and/orvisceral pleura, both of which exhibit characteristics that areconducive to forming a functional small-diameter blood vessel construct.The tissue used for the one or more layers 12 of the luminal graft 10described herein may be derived from any organism, but preferably cellsderived from vertebrates are used. More preferably, cells derived frommammals (e.g., primates, artiodactyls (such as swine and bovine),rodents, etc.) are used.

In at least one exemplary embodiment, the layer(s) 12 of a luminal graft10 comprises glutaraldehyde-fixed pulmonary ligament tissue and/orvisceral pleura. For example, and without limitation, in at least oneembodiment, the one or more layers 12 of luminal graft 10 may comprisepleura tissue and/or pleura ligament tissue, which both exhibitcharacteristics that are conducive to forming a functionalsmall-diameter luminal construct. The tissue used for the one or morelayers 12 of the luminal graft 10 described herein may be derived fromany organism, but preferably comprises cells derived from vertebrates.For the avoidance of doubt, as used herein, the term “pleura tissue”means and includes tissue from the visceral pleura, pulmonary pleura,parietal pleura and/or, more specifically, mediastinal pleura.Furthermore, the term “pleura ligament tissue” as used herein means andincludes tissue from the pulmonary ligament.

For reference, a pleura is a serosa membrane that folds back onto itselfto form a two-layered membrane structure. Generally, the outer pleuralines the thoracic cavity, whereas the inner pleura (visceral orpulmonary pleura) covers the lungs. The parietal pleura lines the innersurface of the chest wall, covers the superior surface of the diaphragmand encases all of the thoracic viscera (excluding the lungs).Accordingly, the parietal pleura separates the pleural cavity (where thelungs are positioned) from the mediastinum or the “middle” section ofthe chest cavity.

The parietal pleura is divided into different portions according to itsposition. For example, the costal pleura is the portion of the parietalpleura that lines the inner surfaces of the ribs and intercostals, thediaphragmatic pleura is that which lines the convex surface of thediaphragm, and the cervical pleura is the portion that rises into theneck and over the apex of the lung. Furthermore, mediastinal pleura isthe portion of parietal pleura that defines the mediastinum and encasesall of the thoracic viscera except for the lungs, as it runstherebetween.

As the mediastinal pleura separates the right and left lungs, inflationof the lungs causes a corresponding expansion of the mediastinalmembrane, thereby resulting in significant friction between themediastinal pleura and the lungs' surfaces during breathing. While themediastinal pleura is relatively thin, it nevertheless exhibitssubstantial integrity and elasticity to accommodate the lungs' expansionand tolerate the friction imposed thereby. The significant elasticity ofthe mediastinal tissue is indicative of its composition, which consistsof multiple fiber-sheet layers having an abundance of elastin fibers inaddition to the collagen typically present in connective tissue. This issignificant from a tissue product and medical application perspectiveas, unlike collagen, elastin cannot be fixed and largely retains itselasticity and biomechanical activity post-fixation. Furthermore, bothsurfaces of the mediastinal pleura are lined with mesothelium, whichprovides for significant integrity, promotes its friction-resistantnature, and provides for antithrombotic properties. Accordingly, postfixation mediastinal pleura has high elasticity, and both sides of themediastinal pleura tissue are smooth and covered with an epitheliallayer that secrets a lubricant.

Visceral pleura plays an important physiologic role in lung function andis responsible for approximately twenty percent (20%) of the work doneduring deflation. It is inherently compliant and non-thrombogenic due,at least in part, to its mesothelial lining. In particular, visceralpleura (as well as the pulmonary ligament, which will be discussed infurther detail below) comprises a smooth, continuous layer ofmesothelial cells, which perform a significant role in maintaininghomeostasis akin to vascular endothelial cells. Furthermore, thepulmonary visceral pleura has an extracellular composition that issimilar to arteries, with a roughly equal proportion of elastin andcollagen. Indeed, while arteries have a collagen to elastin ratio (C/E)ranging from 3.0-1.0, visceral pleura exhibits about a 1.0 C/E ratio(note that because collagen is dominant in most tissue (C/E>>1), a C/Eratio of around 1.0 is considered an elastin dominant tissue). Perhapsmore specifically, the C/E ratio of visceral pleura varies to someextent along the various portions of the tissue even with respect to thesame lung (e.g., front, dorsal, apex, etc.), as well as from baby,adult, to aged swine. As such, at least one representative C/E ratiorange for visceral pleura (including the pulmonary ligament) is fromabout a 1.2 ratio value to about a 0.8 ratio value (all of which areconsidered elastin dominant).

As discussed in further detail herein, visceral pleura grafts do notsuppress in vivo remodeling, but rather their efficacy is, at least inpart, due to the grafts' unique ability to work with the natural in vivoremodeling process to result in a functional vessel graft. Importantly,visceral pleura is abundantly available (e.g., from swine, bovine, andother mammalian sources) and can be easily and quickly fabricated intografts having numerous diameter sizes. Overall, the composition,biocompatibility, and material properties of the pulmonary visceralpleura make it a strong candidate for vessel grafts.

The pulmonary pleura (also known as visceral pleura) covers the lungsand extends to the hilum where it becomes continuous with the parietalpleura. As the anterior and posterior pleura extend below the pulmonaryveins, the two layers of pleura come together to form the inferiorpulmonary ligament or, as termed herein, the pleura ligament. Hence, thepleura ligament is a double layer of pleura that drapes caudally fromthe lung root and loosely tethers the medial aspect of the lower lobe ofthe lung to the mediastinum. However, and importantly, pleura ligamenttissue does not functionally behave the same as two layers of pleura, asthe non-isotropy of pleura ligament tissue is notably different thanjust two layers of pleura. Furthermore, the degree of collagen withinthe pleura ligament is also different than in two layers of pleura, andthe function of the pleura ligament is also different, as pleuraligament tissue resists load in one direction. The pleura ligamenttethers the lung and has substantial elasticity (over 200% extension,which may be a lateral extension) to expand with each inflation of thelung Similar to the mediastinal pleura previously discussed, thesignificant elasticity of pleura ligament tissue stems from its highelastin content, which is beneficial in that it largely retains itselasticity post fixation.

The characteristics of pleura ligament tissue and/or pleura tissue areexceedingly beneficial, especially with respect to luminal grafts 10comprising a small-diameter (≤about 4 mm). Primarily, as previouslyindicated, the major components of pleura ligament tissue and pleuratissue are elastin and collagen, with elastin being the dominantcomponent. This composition is particularly beneficial with respect toforming luminal grafts 10 because elastin is not as prone to fixation ascollagen fibers. Furthermore, where a luminal graft 10 comprises asmall-diameter construct, tissue comprising elastin largely maintainsits elasticity and hence biological mechanical activity followingfixation. Additionally, both pleura ligament tissue and pleura tissueare thin, native tissues that are readily available, easily harvested inlarge sections, and do not require processing prior to formation.

Moreover, and importantly, mesothelial cells cover the surface(s) ofboth pleura ligament tissue and pleura tissue, which provides for aslippery, non-adhesive and protective surface. Specifically, pleuraligament tissue, mediastinal pleura, and parietal pleura havemesothelium on both sides, while pulmonary pleura has mesothelium ononly one side. The mesothelial surfaces of the pleura ligament andpleura tissue have antithrombotic and anti-adhesive properties, whichare especially beneficial in connection with the low-pressure conditionspresent within a small-diameter vessel and for nerve regenerationconduits and applications. In application, where the inner-most layer 12of the luminal graft 10 comprises visceral/pulmonary pleura, the tissueis positioned such that the inner surface 17 of the layer 12 comprisesthe side of the tissue having mesothelium thereon. As pleura ligamenttissue, mediastinal pleura and parietal pleura have mesothelium on bothsides, such considerations are not necessary where the inner-most layer12 of a luminal graft 10 comprises any of the aforementioned.

Due to the beneficial characteristics of pleura ligament tissue and thepleura tissue, luminal grafts 10 comprising pleura ligament tissueand/or pleura tissue make superior scaffold for vascular cells and nervetissue as they do not require any processing prior to formation (such asthe stripping of muscle or treatment with antibiotics). Furthermore, itis not necessary to seed the same with smooth muscle cells orfibroblasts before implantation as is required with other constructs.Additionally, it has been determined that such tissues areanti-thrombotic, anti-adhesive and attenuate the growth of scar tissuedue to their inherent structure.

For example, as can be seen in FIGS. 3A and 3B, such tissues compriseabundant glycocalyx, which covers the apical surface of such tissue'smesothelial cells and promotes intracellular adhesion while concurrentlyinhibiting coagulation and leukocyte adhesion (the tissue in FIGS. 3Aand 3B comprises pulmonary pleura). Indeed, it has been determined thatswine pulmonary visceral pleura, in particular, contains abundantfibrous extracellular matrices containing collagen, elastin,fibronectin, and laminin, as well as integrins binding sites (e.g., RDGpeptide, the active domain of collagen for cell adhesion) and heparinsulfate and sialic acid which are components of glycocalyx. In additionto the benefits described above, such extracellular matrices providechemical cues to stimulate nerve regeneration and the collagen, elastin,and glycocalyx promote Schwann cell migration and proliferation.

Now referring back to the configuration of the luminal graft 10 itself,as previously described, each layer 12 of the luminal graft 10 is shapedto form an elongated tubular construct comprising a seam 20 running thelongitudinal length thereof. The seam 20 comprises the two edges 16, 18of the layer 12 sealed together via one or more closure mechanisms 22.The precise configuration of the seam 20 may be modified depending uponthe particular application of the graft 10 and/or patientspecifications. For example, in at least one embodiment, the first edge16 overlaps the second edge 18 at the seam 20 such that the innersurface 17 of the first edge 16 is secured by the closure mechanism(s)22 to the outer surface 19 of the second edge 18. As shown in FIG. 7,the edges 16, 18 may alternatively be positioned relative to each othersuch that the inner surfaces 17 of both edges 16, 18 are held togetherby the closure mechanism(s) 22 at the seam 20. Still further, the firstand second edges 16, 18 may not overlap at all, but rather be coupledend-to-end via the closure mechanism(s) 22.

The closure mechanism(s) 22 may comprise any mechanism or adhesivecapable of securely coupling the two edges 16, 18 of the layer 12together. Furthermore, in at least one embodiment, the closure mechanism22 is sufficiently flexible or semi-flexible to render the luminal graft10 with appropriate curvature as needed (see FIG. 7).

The closure mechanism(s) 22 may be positioned along the length of theseam 20 in any fashion provided a secure coupling of the first andsecond edges 16, 18 can be achieved and a seal formed along the seam 20.For example, in at least one embodiment, a closure mechanism 22 spansthe entire length of the seam 20. Alternatively or additionally, closuremechanisms 22 may be positioned strategically or sporadically along theseam 20, provided the two edges 16, 18 of the layer 12 are securelysealed together. Still further, the closure mechanism(s) 22 may be usedin conjunction with an adhesive or other biological bonding agent.

Now referring to FIGS. 3A-6B, various embodiments of specific closuremechanism 22 configurations are shown. In the at least one exemplaryembodiment of FIGS. 3A and 3B, the closure mechanism 22 comprises anarrow-lock configuration. Specifically, the layer 12 comprises aplurality of protrusions 24 positioned along the length of the firstedge 16 and a plurality of corresponding openings 26 positioned alongthe length of the second edge 18. The protrusions 24 may be comprised ofany implantable material suitable for use with the luminal graft 10 suchas, by way of non-limiting example, stainless steel, implantablebiopolymers, polytetrafluoroethylene, etc.

The protrusions 24 and openings 26 are configured and sized such thateach opening 26 may easily receive its corresponding protrusion 24 whenthe first and second edges 16, 18 are moved in close proximity to eachother. Additionally, the size and shape of the protrusions 24 andopenings 26 are also configured such that when the protrusions 24 areseated within their respective openings 26, the protrusions 24 cannot beeasily withdrawn. For example, as shown in FIGS. 3A and 3B, theprotrusions 24 of the closure mechanism 22 may comprise an arrow-likeconfiguration such that they can slide easily into an opening 26, butnot be withdrawn therefrom. In this manner, after the protrusions 24 arereceived by the openings 26, the protrusions 24 are securely positionedtherein, thus sealing the first and second edges 16, 18 of the layer 12together along the seam 20.

It will be appreciated that while the protrusions 24 shown in FIGS. 3Aand 3B each comprise an arrow-like configuration, the protrusions 24 maybe shaped in any manner provided they are capable of being received byand securely engaging the corresponding openings 26. Furthermore, it isnoted that the spacing between the protrusions 24 allows for theunderlying luminal graft 10 to remain flexible such that the graft 10may have curvature as needed when used as a replacement vessel or as anerve guidance conduit in a body.

FIGS. 4A and 4B illustrate an alternative embodiment of a closuremechanism 22 of the present disclosure that does not require puncturingor creating a hole in the layer 12 of the luminal graft 10. In the atleast one exemplary embodiment of FIGS. 4A and 4B, the closure mechanism22 comprises a first magnetic strip 28 and a second magnetic strip 30,where the first and second magnetic strips 28, 30 are each configuredfor attachment to opposing edges 16, 18 of the layer 12. Each of themagnetic strips 28, 30 may be comprised of any permanent magneticmaterial known in the art and may be flexible, semi-flexible orarticulated. In at least one embodiment, the first and second magneticstrips 28, 30 each comprise a thin, smooth, ferromagnetic strip.

The first and second magnetic strips 28, 30 of the closure mechanism 22may be configured in any shape provided each strip 28, 30 liesrelatively flat in application. Furthermore, the first and secondmagnetic strips 28, 30 are polarized such that they are magneticallybiased toward each other. Due to the matching configuration and the biasbetween the first magnetic strip 28 and the second magnetic strip 30,the first and second magnetic strips 28, 30 are capable of magneticallyengaging each other. When the first and second magnetic strips 28, 30are magnetically engaged, the two magnetic strips 28, 30 form a singleunit. Accordingly, when the first edge 16 is folded over or moved intothe general proximity of the second edge 18 (see the directional arrowsshown in FIG. 4A), the first and second magnetic strips 28, 30magnetically engage, thereby securely sealing the first and second edges16, 18 of the layer 12 together.

It will be noted that the magnetic strips 28, 30 may be positioned oneither side of the edges 16, 18 of the layer 12. For example, in theleast one embodiment shown in FIGS. 4A and 4B, the magnetic strips 28,30 are positioned on opposite surfaces of the layer 12 (e.g., one oninner surface 17 and one on outer surface 19). In this manner, when thesecond edge 18 is folded over as indicated by the directional arrows inFIG. 4A, the magnetic strips 28, 30 are in direct contact with eachother (see FIG. 4B). Alternatively, the magnetic strips 28, 30 may bepositioned such that when the second edge 18 engages the first edge 16,at least one layer 12 is compressed between the two magnetic strips 28,30 and the magnetic strips 28, 30 magnetically engage each othertherethrough.

Now referring to FIGS. 5A and 5B, an additional embodiment of a closuremechanism 22 of the present disclosure is shown. In this at least oneembodiment, each of the edges 16, 18 of the layer 12 further comprise aseries of perforations 34 extending along at least part of the lengththereof. The perforations 34 are configured and spaced to allow forsutures 36 to be threaded therethrough as shown in FIG. 5B. In at leastone embodiment, the perforations 34 are formed through the layer 12itself. Alternatively or additionally, the closure mechanism 22 maycomprise two perforated strips (not shown), each coupled with an edge16, 18 of the layer 12 such that the underlying tissue is not puncturedby the sutures 36 threaded therethrough. In at least one embodiment,each of the edges 16, 18 of layer 12 comprise both a series ofperforations 34 formed therethrough and a perforated strip such that adouble perforated line extends substantially the entire length of theseam 20.

The sutures 36 may comprise any material suitable for use in connectionwith the luminal graft 10 including, without limitation, metal wire,wound and braided wires, suture material, plastic strings, rope and thelike. As shown in FIG. 5B, the sutures 36 may be performed to include asingle revolution through paired perforations 34 on the first and secondedges 16, 18 thereby defining a loop and securing the seam 20. In thisat least one embodiment, the loops are positioned perpendicular to thelongitudinal axis of the seam 20. It will be appreciated that the suture34 and its resulting loops are flexible and may be adjusted in shape,size and orientation based on how the suture 34 is tensioned, thesuturing technique employed and/or the diameter(s) of the underlyingluminal graft 10 (e.g., pursuant to certain principles of vascularsurgery, the smaller the diameter of the graft, the smaller the suture).Accordingly, the underlying luminal graft 10 may be flexible or rigiddepending upon the type of suture and suturing technique employed.

FIGS. 6A and 6B illustrate yet another embodiment of a closure mechanism22 of the present disclosure. In this embodiment, the closure mechanism22 comprises a series of clips 38 positioned along the length of theseam 20 of the luminal graft 10. Each clip 38 of the closure mechanism22 is configured and sized to securely compress the edges 16, 18 of thelayer 12 together when such edges 16, 18 are positioned within the clip38 and the clip 38 is deployed. For example and as shown in thecross-sectional view of FIG. 6B, in application, the two edges 16, 18 ofthe layer 12 are brought together to form seam 20 and the clips 38 areslid thereover. The clip 38 may then be compressed or deployed, therebysealing the edges 16, 18 of the layer 12 together. The clips 38 maycomprise any configuration, provided they are comprised of a materialsuitable for use in connection with the luminal graft 10 and cansecurely engage the edges 16, 18 of the layer 12 and seal the seam 20.

FIG. 7 illustrates a perspective view of at least one embodiment of aluminal graft 10 according to the present disclosure that issufficiently flexible to render the graft 10 with curvature. In thisembodiment, the luminal graft 10 comprises one layer 12 that is sealedalong the seam 20 with a closure mechanism 22 comprising sutures. Asshown in FIG. 7, the graft 10 is configured such that the first andsecond edges 16, 18 of the layer 12 are secured in a manner so that theinner walls 17 of both the first and second edges 16, 18 are in contactwith each other.

Due to its unique properties, a luminal graft 10 of the presentapplication is particularly suitable and exceedingly functional for manydifferent medical applications. Primarily, the luminal grafts describedherein do not experience the problems associated with conventionalluminal constructs such as stenosis, aneurysm or thrombosis formation.Because of this, the luminal grafts 10 are capable of maintaininglong-term patency, an undertaking that has not been previously achievedin conventional constructs. Indeed, in at least one experiment conductedusing embodiments of luminal graft 10, the grafts 10 were fullyfunctional at 24 weeks following coupling with a murine femoral arteryand maintained their patency for 6 months (subject grafts formed oflayers of pulmonary pleura and pleura ligament tissue).

Additionally, the luminal grafts 10 of the present disclosure are alsocapable of arterializing over time. For example, an embodiment ofluminal graft 10 was prepared using as graft of femoral artery of rat.Surprisingly, at the 24th week following implantation, the subjectluminal graft not only contracted efficiently in response to 60 mM ofpotassium chloride (about 10 mmHg elevation), but also achieved about70% of maximal vasodilation in response to an endothelium-dependentvasodilator (acetylcholine, ACh). Furthermore, vasodilation in responseto a nitric oxide donor showed definite vascular tone, and significantEndothelin1 (ET1)-induced contraction was achieved. Accordingly, allfunctional measurements indicate that the luminal graft had arterializedand was functioning as a true artery. Histological images confirmed thata substantial smooth muscle media developed (as shown in FIGS. 15A and15B) adjacent to the graft and endothelium covered the luminal surfaceat this time point (as shown in FIGS. 15B and 12C). The external spaceof the graft was occupied by connective tissue which combined with thegraft to act as the adventitia of the new artery (FIG. 15A).

In light of this, in at least one application, embodiments of theluminal graft 10 may be employed as an arteriovenous (AV) graft for usein connection with the delivery of hemodialysis. An AV graft is createdby connecting an artery to a vein with a tube of biocompatible material,and implanting the tube subcutaneously. The AV graft then functions asan artificial vein that can be used repeatedly for needle replacementand blood access. A high occurrence of thrombosis has historically beenassociated with this procedure, which typically develops due to poorblood flow therethrough. Furthermore, aneurysms and graft failure areadditional common complications of conventional AV grafts inhemodialysis patients.

The luminal graft 10 of the present disclosure is suitable for use as anexemplary AV graft due to its unique ability to arterialize and avoidthe complications typically associated with conventional grafts.Specifically, because the luminal graft 10 is capable ofarterialization, the luminal graft 10 is capable of maintaining adequateblood flow through the vascular access point when employed as AV graft,thereby reducing the likelihood of thrombosis. Additionally, the intimalwall of the luminal graft 10 is an anti-thrombotic material that doesnot produce stenosis or aneurysm. Accordingly, application of theluminal graft 10 of the present disclosure as an AV graft forhemodialysis overcomes many of the shortcomings of conventional AVgrafts.

Yet another exemplary application of the luminal graft of the presentapplication relates to peripheral nerve regeneration techniques (forexample, tubulization). As previously described, although it is widelyaccepted that the peripheral nervous system is capable of regenerationin a laboratory setting, current strategies are associated with quitesignificant limitations in a patient's functional recovery. Indeed, allautologous nerve graft alternatives (including decellularized nervegrafts and autologous and non-autologous conduits) demonstrate somedegree of efficacy, but only when used with sensory nerves having smallgaps (<3 cm). There is a significant need for nerve guidance conduitscapable of repairing large gaps.

Generally, with respect to the peripheral nervous system, there are fourrequirements for an “ideal” tissue-engineered nerve graft: 1) that it iscompatible with the surrounding tissues; 2) that it is of an adequatesize and length; 3) that it contains substances that enable and supportaxonal regeneration of the nerve; and 4) that it protects theregeneration of the nerve fibers from scar invasion. Generally,biological tissue is better on compatibility as compared to artificialtissue. In the graft, complete compatibility of pleura tissue with thehost was found as there was no evidence of inflammation or rejection ofthe graft. Here, due to the composition and configuration of the luminalgraft 10 and, perhaps more specifically, the use of swine PVP which isacellular and contains both collagen and elastin, the intimal wallsthereof are anti-adhesive, provide protection, and attenuate the growthof scar tissue. Accordingly, the luminal graft 10 contains substancesthat enable and support axonal regeneration of the nerve and is capableof protecting the regenerating nerve fibers from scar invasion when usedas a nerve guidance conduit.

Furthermore, the luminal graft 10 can be manufactured to variousdiameters and lengths depending on the particular application and/ornerve(s) at issue. In this manner, the luminal graft 10 can be formedinto a nerve guidance conduit having a size and length tailored to thenerve lesion/defect at issue (see FIGS. 14A-14C). Finally, the materialsthat form luminal graft 10 (namely, pleura ligament tissue and/or pleuratissue) are biocompatible, provide sufficient flexibility to achieve anoptimal nerve regeneration environment, and do not cause nervecompression.

Consequently, it is apparent that the characteristics of the luminalgraft 10 and its underlying materials satisfy all major requirements ofa nerve regeneration conduit. Furthermore, experimental results stemmingfrom tests of the luminal graft 10 in this capacity (described infurther detail below) showed that the resulting nerve guidance conduitis capable of maintaining long-term patency and even recoversneuromuscular functions.

Accordingly, use of pleura ligament tissue and/or pleura tissue in thecomposition of the luminal graft 10 enables the graft 10 to be effectivein the aforementioned applications in large part because it is capableof maintaining long-term patency. With respect to blood vessels, thedisclosure of the present application provides for a luminal graft 10suitable for the replacement of blood vessels, including, withoutlimitation, small-diameter blood vessels. Use of pulmonary ligamenttissue and/or visceral pleura in the composition of luminal graft 10enables the graft 10 to be effective as a functional small-diameterconduit vessel (for vascular or nerve applications, for example) capableof maintaining long-term patency. Because of the mesothelial surfaces ofglutaraldehyde-fixed pleura ligament tissue and pleura tissue, as wellas the other beneficial properties of such tissues described herein,manufacturing these tissues into scaffolds provides a graft environmentsuitable for the infiltration, adhesion, proliferation and expressionand maintenance of cells so that the scaffolds are replaced with tissuemade of vascular and/or nerve cells, depending on the application.

In at least one exemplary embodiment especially suited to promoteneuronal regeneration for large gaps in nerve injuries, the luminalgraft 10 hereof may also comprise one or more intraluminal microchannels1602. As shown the cross-sectional view of a layer 12 of FIG. 16A, theinner surface 17 of a layer 12 of the graft 10 may comprise or define aseries of longitudinally positioned, intraluminal microchannels 1602.Such microchannels 1602 may comprise a series of ridges or other detentsformed on the inner surface 17 of a layer 12, or may be defined by aplurality of degradable microspheres 1604 positioned thereon inlongitudinally extending rows (see FIG. 2B). In such embodiments, themicrospheres 1604 may be aligned and affixed on a layer 12 as parallelcohorts (affixed, for example, using fibrin sealant). It will beappreciated that only the inner-most layer 12 of a graft 10 may comprisethe microchannels 1602 or, alternatively, where a graft 10 comprisesmore than one layer 12 (whether rolled or stacked from different sheetsof tissue), more than one (or all) of the layers 12 may comprisemicrochannels 1602 on each of their respective inner surfaces 17.

The microspheres 1604 themselves may be made of natural and/or syntheticmaterials. For example, in at least one embodiment, one or moremicrospheres 1604 may be formed from biological polymers (alginate,chitosan, collagen, and fibrin) and/or synthetic polymers (poly lacticacid (PLA), poly-glycolic acid (PGA), and poly lactic-co-glycolic acid(PLGA)). Furthermore, the microspheres 1604 may be bio-absorbable and/orbiodegradable. In at least one exemplary embodiment, the microspheres1604 are formed from PLGA, which is biocompatible and biodegradable,exhibits a wide range of erosion times, has tunable mechanicalproperties and, importantly, is an FDA-approved polymer.

As shown in FIG. 16B, where a graft 10 comprises multiple rolled orstacked layers 12 that define microchannels 1602 on their inner surfaces17, the microspheres 1604 (or other ridges or the like) form pillarsbetween the various layers 12 thus defining microchannels 1602 (space)therebetween. Here, the portion of the graft 10 shown comprises a firstlayer 12 ₁ having longitudinal microchannels 1602 formed frommicrospheres 1604, positioned below a second layer 12 ₂ havinglongitudinal microchannels 1602 formed from microspheres 1604,positioned below a third layer 12 ₃. When multiple layers 12 ₁, 12 ₂, 12₃ are used to form the graft 10 (whether using multiple sheets of tissueor through a rolled configuration), each microchannel 1602 formedbetween two layers comprises four walls—i.e. sidewalls formed by themicrospheres 1604 (or ridges or the like) and a ceiling and floor formedby the bases of layers 12 themselves (here 12 ₁, 12 ₂ and 12 ₃).

The distances between the aligned cohorts of microspheres 1604 (and/orridges or the like) and the diameter of the microspheres 1604/ridgesdetermine the cross area (and ultimately number) of the microchannels1602. It will be appreciated that the dimensions of the microchannels1602 and/or microspheres 1604/ridges are fully customizable, need not beconsistent throughout an entire layer 12 and/or the luminal graft 10,and may be flexibly adjusted based on the desired application.Microsphere 1604 dimensions may even be adjusted on a per-sphere basis.Furthermore, the number of microchannels 1602 present on a layer 12 maybe determined by microsphere 1604/ridge/etc. diameter, the distancebetween two alignments of microspheres 1604/ridges/etc., the thicknessof the tissue forming the layer 12 itself, and the cross-sectional areaof the graft 10.

In at least one embodiment, the microsphere 1604 diameters may comprise100, 200, and/or 400 μm to build up four types of microchannels 1602having cross areas of about 100×100 μm², 200×200 μm², and 400×400 μm².In another embodiment, the number of microchannels 1602 present will beup to about 150 when the cross area of a microchannel 1602 is around200×200 μm² and the diameter of the luminal graft 10/nerve guidanceconduit is 3 mm. Still further, in certain cases, microchannels 1602comprising greater than about 20 μm may be desirable for guiding nerveregeneration due to debris in proximal and distal nerve stumps andfibroblasts migration during nerve regeneration. It will be appreciatedthat no limitation is intended by providing such dimensional examples.Indeed, as previously stated, the dimensions and number of themicrochannels 1602 and/or microspheres 1604 of a luminal graft 10 arefully customizable and may be selected as desired.

In application and as shown in FIG. 16C, the microchannels 1602 providetopographic intraluminal guidance cues, while the extracellular matrixof the tissue of the layers 12 (e.g., swine PVP) provide chemical cues,both of which promote the regeneration of axons 1610. Without theformation of an aligned extracellular matrix bridge within a hollowconduit, limited migration of native Schwann cells on the side of thelesion typically occurs (i.e. hollow conduits are limited in theirability to guide regenerating axons as they lack a scaffold across whichregeneration can occur). However, each microchannel 1602 serves as astructural guidance conduit, forming a sub-lumen that mimics the naturalfascicular organization of a nerve. In this manner, the microchannel1602 configuration of the luminal graft 10 can significantly enhancenerve regeneration therethrough. However, even where the luminal graft10 comprises cohorts of microspheres 1604/ridges and Schwann cells beginto proliferate within the microchannels 1602, the growth factorssecreted by such Schwann cells alone may be not be sufficient tostimulate axonal sprouting. To address this, in at least one exemplaryembodiment of the present disclosure, exogenous neurotrophic factors areencapsulated within one or more of the microspheres 1604 such that thehydrolysis of the microspheres 1604 during biodegradation/absorptionresults in the controlled release of the encapsulated substance (i.e.neurotrophic factor(s)) over time. Additionally, as the microspheres1604 degrade over time, so too will the microchannels 1602 definedthereby, thus allowing for the more spacious accommodation of nervefiber expansion.

Generally, in nerves, neurotrophic or growth factors can enhancefunctional regeneration by supporting axonal growth, Schwann cellmigration and proliferation, and/or increasing neuroprotection throughreceptor-mediated activation of a specific intrinsic signaling pathway.Neurotrophins, including without limitation nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), andneurotorophin-4/5 (NT-4/5), promote nerve regeneration. NGF is presentat low concentrations in healthy nerves, but is upregulated followinginjury in the distal nerve stump and plays an important role in thesurvival of sensory neurons and neurite outgrowth. NGF (acting throughthe high affinity TrkA receptor) may improve histological,electrophysiological, and functional outcomes, at least in small animalmodels of nerve gap injury, and the application of exogenous NGF hasbeen linked to increased sensory neuron regeneration. NT-3 promotesmotor neuron survival and promotes the axonal growth of both motor andsensory neurons.

Conventionally, however, the application of neurotrophic factors has notproven effective, especially in promoting nerve regeneration in responseto a nerve gap injury. This may be attributed—at least in part—to poorrelease kinetics because most conventional delivery systems exhibit ahigh initial burst release. Indeed, it remains difficult to deliver theneurotrophic factors using microspheres over the entire duration ofnerve regeneration due to the relatively small capacity of themicrospheres themselves.

Encapsulation of one or more neurotrophic factors within a microsphere1604 of the luminal graft 10 allows for the application of one or moreneurotrophic factors in combination with or via sustained releasedelivery systems or scaffolds to further enhance axonalregeneration—particularly for conduits used with nerve gap injuries.Indeed, this, taken in conjunction with the use of pleura ligamenttissue and/or pleura tissue to form the layer(s) 12 of the graft 10 andthe use of microchannels 1602 thereon, allow for the successfulcombination of multiple stimuli, all of which promote axonalregeneration for even larger gaps in nerve injury. The placement of eachmicrosphere 1604 on the layer 12, the substance that each microsphere1604 contains, and the speed at which each microsphere 1604 dissolvesmay all be manipulated to achieve the controlled release of particularsubstances to the regenerating axons in a controlled manner (i.e. toachieve spatially-specific and/or temporal-specific release) and matchthe Schwann cells' migration and proliferation through the microchannels1602 of the luminal graft 10.

Primarily, temporal-specific release from the microspheres 1604 may beachieved by using at least two different types of microspheres 1604,each having a different degradation rate for timing the release of theencapsulated substances therefrom. In other words, the biodegradabilityof the microspheres 1604 is controllable so that the release ofneurotrophic factors can occur at two different time points (e.g., firstrelease at 7 days and second release at 21 days, which will be adjustedfor longer grafts 10 of 6 cm). Furthermore, the microspheres 1604 may bespatially arranged on the one or more layers 12 to leverage thisvariable release timing to facilitate desirable results—such asreleasing neurotrophic factors or other agents at specified locationswithin the luminal graft 10 to match the cell migration andproliferation through the microchannels 1602.

For example, in at least one nerve regeneration application as shown inFIGS. 16C and 17, the microspheres 1604 with shorter biodegradableperiods are positioned on both the proximal and distal portions of thelayer 12/luminal graft 10 where they are connected to the proximal anddistal stumps of the injured nerve (see axonal sprout 1610 in FIG. 16Cand the areas marked A and D in FIG. 17). Conversely, the microspheres1604 with longer biodegradable intervals are positioned in the middleportion of the layer 12/luminal graft 10 (see areas marked B and C inFIG. 17, for example).

This delayed release of the substances within the middle microspheres1604 allows for the Schwann cells 1610 growing through the microchannel1602 to reach that area of the layer 12 prior to or concurrently withthe substances being released. Indeed, using these design principles,the release of the substances from the microspheres 1604 may becoordinated to roughly match the time that the Schwann cells migrateinto such areas (i.e. temporal-specific release). In this manner, thelate release of the encapsulated substances/neurotrophic factors caneffectively match and stimulate Schwann cell proliferation continuouslythrough the microchannel 1602 to fill the entire conduit/microchannel1602.

It is also contemplated that different layers 12 of the luminal graft 10may comprise microspheres 1604 having different degradation rates. In atleast one embodiment where the luminal graft 10 comprises threeconcentrically wrapped layers 12, the innermost layer 12 may comprisemicrospheres 1604 having the fasted degradation (release) rate, followedby the microspheres 1604 of the middle layer 12, while the microspheres1604 of the outer layer 12 degrade the slowest. Accordingly, the conceptof varying degradation rates of the microspheres 1604 to achieve acontrolled release of substances encapsulated therein may be employed inany manner to achieve the effect desired.

In addition to varying the degradation rates of the microspheres 1604,the substance(s) encapsulated within a microsphere 1604 and itsplacement on the layer 12 may also be manipulated to achieve certaingoals. As shown in FIG. 17, it may be desirable for the microspheres1604 encapsulating a first neurotrophic factor to be distributed on theproximal half (marked A and B) of the layer 12 and the microspheres 1604encapsulating a second neurotrophic factor to be distributed on thedistal half of the layer 12 (marked C and D). In this manner, thetechnique provides the capability for spatially-specific release of anencapsulated substance. Notably, this spatially-specific release may becustomized to achieve a desired effect.

In at least one exemplary embodiment, the proximal half (marked A and B)of the layer 12 comprises PLGA microspheres 1604 encapsulating NGF andthe distal half of the layer 12 (marked C and D) comprises PLGAmicrospheres 1604 encapsulating NT-3. In application to nerveregeneration, these neurotrophic factors will be released and anastomoseto proximal nerve stumps upon degradation and subsequent rupture of themicrospheres 1604. NGF and NT-3 release stimulates Schwann cellmigration and proliferation in the microchannels 1602, and thedegradation of the PLGA microspheres 1604 also provides more space forthe Schwann cells to grow into. The degradation of the PLGA microspheres1604 may also be controlled at an incremental delay axially from ends tomiddle of the luminal graft 10 as previously described in connectionwith the temporal-specific release approaches described herein. There,the release of NGF from the microspheres 1604 placed within the proximalhalf of the layer 12 will be sequentially delayed from proximal end tomiddle of the layer 12. The release of NT-3 from the microspheres 1604placed within the distal half of the layer will be sequentially delayedfrom distal end to middle of the layer 12 to stimulate Schwann cellmigration from the distal stump of the injured nerve to middle of theluminal graft 10. Because NT-3 may delay axonal degeneration, the NT-3release in the distal half of the luminal graft 10 is designed to delayWallerian degeneration. Therefore, the proposed luminal graft 10 canbridge a large peripheral nerve defect (for example, up to 6 cm).

The combination of neurotrophic/growth factors can be easily adjustedusing the present design and any number of different substances may beemployed as desired within the microspheres 1604. Accordingly, bymanipulating where and when particular neurotrophic factors or othersubstances are released, a clinician can use the luminal graft 10 toprovide specific therapies tailored to a specific patient and/or theapplication at issue.

Use of the presently disclosed luminal grafts 10 that incorporatebiomolecular therapy, delivery technique, and intraluminal structure toadvance a potential “off-the-shelf” peripheral nerve guidance conduitfor the repair of large nerve injury (e.g., >4 cm). The embodiments ofthe luminal graft 10 presented herein are abundant in extracellularmatrix (elastin, collagen and glycocalyx), intraluminal microchannels1602, and spatial and temporary release options for neurotropic factorsand/or other substances, the combination of which can effectivelypromote axonal regeneration for both small and large gaps in nerveinjury.

Application of the luminal graft 10 may be particularly useful topatients who have been diagnosed with peripheral artery disease,end-stage renal disease (ESRD), or who have undergone coronary bypasssurgeries or suffered peripheral nerve damage. The devices, systems andmethods of the present disclosure can be used in connection with suchpatients to provide a safe, effective, and long-term solution wheresmall-conduit or other vessels may be compromised or damaged.

Now referring back to FIGS. 8A-8D, an exemplary system 200 formanufacturing a luminal graft 10 of the present disclosure is shown. Asthis system 200 is designed to assemble a luminal graft 10, system 200comprises and, in application produces, many of the same components ofluminal graft 10. Accordingly, like reference numerals depict likecomponents throughout the Figures of this disclosure.

FIG. 8A shows an exemplary embodiment of at least a portion of a system200 for manufacturing a luminal graft 10. In at least one embodiment,the system 200 comprises a mandrel 100 and one or more layers 12 oftissue. Each of the layers 12 of tissue comprises a closure mechanism 22configured to couple therewith and seal the resulting seam 20. Theclosure mechanism(s) 22 may or may not be pre-attached to the layer(s)12 in locations conducive to the creation of a desired seam 20 and alumen 14 comprising the appropriate diameter D.

The mandrel 100 comprises a cylindrical configuration having a diameterd. The specific configuration and dimensions of the mandrel 100 may bemodified, depending on the desired design of the resulting graft 10and/or particular aspects of such graft's components. For example, in atleast one embodiment where a varying diameter luminal graft 10 isdesired, the diameter d of the mandrel 100 may gradually increase ordecrease along the length of the mandrel 100.

Each layer 12 of the system 200 comprises a relatively flat sheet offixed tissue comprised of any tissue type suitable for the manufactureof a luminal graft 10. In at least one exemplary embodiment, thelayer(s) 12 comprise one or more sheets of pulmonary ligament tissue orvisceral pleura. Alternatively or additionally, the layers 12 of a graft10 may each be comprised of a different type of tissue or even asynthetic material or scaffold. However, even where multiple types oflayers 12 are employed in the construct of a luminal graft 10, theinner-most layer 12 of the luminal graft 10 comprises a sheet ofpulmonary ligament tissue or visceral pleura such that mesothelium linesthe luminal surface of the graft 10.

Where one or more of the layers 12 of a graft 10 comprise tissue, thetissue to be used for the layer(s) 12 of the graft 10 must be harvestedand prepared prior to use. Accordingly, such layer(s) 12 may bepurchased pre-processed in accordance with the desired specifications orthe harvesting and preparation may be performed directly. If necessary,harvesting and preparation may be performed as is known in the medicalarts and as is appropriate with respect to the type(s) of tissues used.Where the layer(s) 12 of tissue comprises pulmonary ligament tissueand/or visceral pleura, such tissue may be harvested and prepared as setforth in International Application Number PCT/US2013/025591 to Kassab etal., which is incorporated herein by reference in its entirety, anddescribed generally below (at least in part).

For general reference, an overview of a pulmonary ligament and/orvisceral pleura harvesting procedure is described as follows. In atleast one method, targeted biological tissues are pre-stretched orotherwise pre-stressed in vivo for optimal function prior to harvesting.Thereafter, the tissue is extracted from a mammal and placed in arelatively cold saline solution to promote preservation. For example,such tissues may be isolated via blunt dissection from an en bloc lungobtained from a local abattoir and subsequently stored unstretched in0.25% buffered glutaraldehyde for about 48 hours at 23° C. At or beforethe time of processing, the tissue can be inspected for bloodinfiltration, fatty material, perforations, and/or other irregularities,and portions of the tissue containing the same can be treated to eitherremoved the undesired components or discarded/disregarded in view ofother portions of the tissue that are relatively homogenous and free ofundesired properties, such as perforations or fat.

After the desired portions of the tissue are selected from the overallresected tissue, the selected membranes are mounted to remove wrinklesand prevent shrinkage and/or folding during fixation and subsequentlysubmerged in a fixation solution. In at least one exemplary embodiment,glutaraldehyde, for example, at 0.25%, 0.625%, 0.625%, etc., is used forfixation/crosslinking collagen, fibronectin, laminin, and glycocalyx ofthe tissue, as applicable, noting that elastin is not significantlycrosslinked by glutaraldehyde. In addition to its crosslinkingcapabilities, glutaraldehyde can also inhibit the immunogenicity of thetissue.

In at least one embodiment, the tissue is submerged in the fixationsolution for about 24 hours. In at least one embodiment, prior tomounting and/or fixation, or after mounting and/or fixation if desired,the pleura ligament and/or pleura tissue may be pre-seeded to make itmore likely to endothelialize. As pleura ligament tissue, mediastinalpleura, and parietal pleura all have mesothelium on both sides andpulmonary pleura has mesothelium on only one side, pre-seeding (alsoreferred to as an endothelial seeding) could be performed on thenon-mesothelial side of the tissue. It will be appreciated that whilethe seeding process may be performed in preparation for the manufactureof a luminal graft 10, this step is optional due to the configuration ofthe graft 10 and the properties of the underlying pleura ligament and/orpleura tissue.

After fixation, a relatively flat piece of fixed tissue will result.Various sizes and/or thicknesses of processed lung ligament and/orprocessed visceral pleura tissues can be tailored for specificapplications. For example, as is described in additional detail below,the tissue can be dissected into a rectangular sheet of appropriatedimensions and wrapped around a cylinder (e.g., 1.6 mm in diameter) forsuturing or otherwise fixing the shape of the graft 10. Additionally, asit has been previously described that the overall thickness of the wallof a luminal graft 10 may be modified based the number of concentriclayers 12 used in the manufacture thereof, the thickness of the graft 10wall may additionally or alternatively be manipulated by using layer(s)12 having a specific thickness. Accordingly, these grafts 10 can be madein a variety of dimensions and/or configurations that are suitable fornumerous grafting applications, including coronary artery bypassgrafting procedures.

In application, the mandrel 100 forms a model over which the layer(s) 12of tissue are wrapped as shown in FIGS. 8A and 8B. When the layer(s) 12are wrapped over the mandrel 100 and the seam 20 is sealed throughdeploying/engaging the closure mechanism(s) 22, the resulting graft 10comprises a configuration similar to the shape of the mandrel 100 (seeFIG. 8C). Most notably, the lumen 14 of the resulting graft 10 comprisesa diameter D that is equivalent to the diameter d of the mandrel 100.Where the system 200 comprises more than one layer 12, the process ofwrapping the layer 12 around the mandrel 100 and deploying the closuremechanism(s) 22 is repeated for each subsequent layer 12. It will beappreciated that the closure mechanism(s) 22 need not comprise the sameconfiguration for each layer 12 of the system 200 and, in fact, may evencomprise different configurations along the same seam 20.

Now referring to FIG. 9, a flow chart is shown representing a method 300for manufacturing a luminal graft 10 in accordance an exemplaryembodiment of the present disclosure. FIGS. 8A-8D will also be used forreference in describing the various steps of method 300.

In at least one embodiment, at step 302, the lumen 14 of the luminalgraft 10 is formed as shown in FIG. 8. Specifically, a first layer 12 iswrapped around the mandrel 100 such that the first and second edges 16,18 of the layer 12 are positioned adjacent to each other and the mandrel100 is substantially encased in the layer 12. As previously discussed,when the layer 12 comprises pleura ligament tissue, mediastinal tissue,parietal tissue or any other pleura tissue having mesothelium on both ofits sides, it is irrelevant as to which side of the layer 12 facestoward the mandrel 100. However, where the layer 12 comprises pulmonarypleura or any other type of pleura tissue having mesothelium on only oneof its sides, at step 302, care should be taken to position the layer 12relative to the mandrel 100 such that the mesothelium side of layer 12faces the mandrel 100 (and thus forms the interior wall or luminalsurface of the luminal graft 10). In this manner, the luminal graft 10is configured such that the mesothelium coats the inner surface 17 (i.e.luminal surface) of the graft 10 and provides an exemplary scaffold forvascular and/or nerve cells along with its non-thrombogenic properties.

In the embodiment shown in FIG. 8A, the closure mechanism 22 comprisesmagnetic strips that were pre-attached to the layer 12. In such anembodiment, after the layer 12 is wrapped around the mandrel 100 at step302, the method 300 proceeds directly to step 304. However, in the eventthe closure mechanism 22 was not pre-attached to the layer 12, method300 proceeds from step 302 to step 302 a prior to advancing to step 304.At step 302 a, the appropriate closure mechanism(s) 22 is/are attachedto the layer 12 in the designated position(s).

At step 304, the closure mechanism(s) 22 of the layer 12 is/arepositioned for deployment. For example, as shown in FIG. 8B, the innersurface 17 of the first edge 16 is positioned adjacent to the innersurface 17 of the second edge 18 such that the first and second magneticstrips are aligned. At step 306, the closure mechanism(s) 22 are engagedand otherwise deployed, thereby forming seam 20 along the length of thegraft 10 and sealing the construct. FIG. 8B illustrates system 200 atstep 306 of method 300, where the closure mechanism 22 comprises twomagnetic strips. Likewise, FIG. 8C illustrates system 200 at step 306 ofmethod 300 where the closure mechanism 22 comprises perforated stripsand sutures.

At step 308, any extra tissue is trimmed away from the exterior of thegraft 10 adjacent to the seam 20, thereby minimizing the profile of thesame. FIG. 8D shows a close-up view of a seam 20 where the extra tissuehas been trimmed away.

In the event the luminal graft 10 comprises a multi-layered construct,following step 308, the method 300 returns to step 302. Accordingly, thesecond layer 12 is wrapped around the previously wrapped layer(s) oftissue 12 and the underlying mandrel 100. As previously described, anyadditional layers 12 (other than the inner-most layer 12) can comprisethe same or different types of tissue and/or synthetic materialsdepending on the desired specifications for the luminal graft 10.Thereafter, the method 300 advances through the steps as previouslydescribed for each of the remaining layers 12 (e.g., if the luminalgraft 10 comprises three layers 12, step 302-308 will be repeated threetimes). Furthermore, in at least one embodiment, step 302 mayadditionally comprise aligning any subsequent layers 12 of the luminalgraft 10 relative to the previously wrapped layer(s) 12 such that theseams 20 of adjacent layers 12 are offset. For example, as shown in FIG.2, placement of the seams 20 on the luminal graft 10 may be varied toensure the construct is sealed and to prevent leaks.

When the final (i.e. outer) layer 12 has been secured at step 308, themethod 300 advances to step 310. At step 310, the mandrel 100 isslidingly removed from the lumen 14 of the luminal graft 10, thusresulting in a luminal graft 10 having a diameter D and configurationdictated by the underlying mandrel 100. At optional step 312, theluminal graft 10 may be stored in about 0.25% buffered glutaraldehydeuntil implantation, and at optional step 314, the graft 10 may be rinsedrepeatedly (for example, five (5) times) with saline and incubated in 50U/ml heparin saline for about thirty (30) minutes prior to implantation.Implantation of the resulting graft 10 may follow the standardtechniques of vascular surgery for anastomosis or arteriovenousgrafting, microsurgery for nerve guidance conduits, or other applicablemethods as appropriate.

In application, at least certain embodiments of the grafts 10 of thepresent disclosure utilize the natural remodeling process of the host toturn passive grafts 10 into functional vessels. Unlike severalconventional approaches that attempt to stymie the remodeling process(e.g., drug elution), the grafts 10 and techniques hereof exploitnatural remodeling to orchestrate the engineering of a new vessel. Inparticular, the grafts 10 can develop into functional conduits in acoronary revascularization setting, even under the complex geometries ofcoronary arteries in motion. Indeed, following several in vivo and invitro studies, certain small-diameter grafts 10 (some with diameters≤1.0 mm) were even patent at 6 months post-implantation, with functionalendothelial and smooth muscle cells. Details of such tests andvalidation processes will now be detailed below; however, it will beunderstood that such studies and findings are included for explanatoryand validation purposes only and are not intended to be limiting in anyway.

Now referring to FIG. 18, when a luminal graft 10 comprisingmicrospheres 1604 is manufactured, each layer 12 is coated with themicrospheres 1604 such that the microspheres 1603 are aligned andaffixed to the layer 12 in parallel cohorts. The layer(s) 12 coated withthe parallel lines of microspheres 1604 is/are then assembled into aluminal graft 10 to form the microchannels 1602.

In at least one embodiment, the alignment of the microspheres 1604 andtheir subsequent affixation to the layer 12 is facilitated by a gratingmodule 1800. FIG. 18 shows a schematic of at least one embodiment ofsuch a grating module 1800. The grating module 1800 comprises a seriesof notches 1802 separated by intervals 1804, which may be used toaxially align microspheres 1604 in preparation for affixing themicrospheres 1604 to a layer 12 of the graft 10. The grating module 1800itself may be manufactured using etching techniques.

The dimensions of each notch 1802 and interval 1804 are selected toproduce the desired microchannel 1602 dimensions on the layer 12 whenthe microspheres 1604 are affixed thereto. Optionally, the depths of thenotches 1802 may be somewhat smaller than their respective widths tofacilitate the affixation of the microspheres 1604 to the layers 12(i.e. so the microspheres 1604 are raised up a bit above the top of eachinterval 1804). For example, in at least one embodiment, the dimensionsof the notches 1802 comprise 100, 200, 400 μm in width and 80, 160, 320μm in depth, respectively, and 6 cm in length (here the notch depths are20% smaller than the notch widths). Such dimensions can accommodatemicrospheres 1604 having diameters of 100, 200, 400 μm, respectively.Furthermore, in such example, the intervals 1804 of the notches 1802, aparameter that determines the dimensions of the microchannels 1802, are100, 200, and 400 μm, respectively. Notably, the dimensions of theintervals 1804 and notches 1802 are independent of the diameters of themicrospheres 1604 and the cross figuration of the microchannels 1602 canbe adjusted by changing the dimensions of the intervals 1804.Furthermore, while specific examples of grating module 1800 dimensionshave been provided, it will be appreciated that it shall not be limitingand any dimensions (and even various dimensions within the same gratingmodule 1800) may be employed.

In application, the microspheres 1604 are loaded into the notches 1802of the grating module 1800 (see FIG. 19), which forms one-by-one rows ofmicrospheres 1604. Where microspheres 1604 having varying degradationrates and/or encapsulating various substances are employed, themicrospheres 1604 are positioned on the grating module 1800 according tothe desired degradation duration (e.g., microspheres 1604 having fasterdegradation rates (early rupture—shown in FIG. 19 as light) at theproximal and distal ends, and microspheres 1604 having slowerdegradation rates (late rupture—shown in FIG. 19 as dark) in the middleportion of the grating module 1800) and/or positioning of where thesubstances should be released within the resulting microchannels 1602.The loading of the microspheres 1604 into the grating module 1800 may beperformed manually or, in at least one embodiment, by a robot. Forexample, a robot for biological 3D printing (e.g., Regenova 3D printer)can be employed to precisely distribute various microspheres 1604 attarget positions on the grating module 1800.

Likewise, a rectangular sheet 1902 of PVP or other desired material thatwill form one or more layer(s) 12 is trimmed to a designed length (e.g.,7 cm) and width (e.g., 1.2 cm for 1 mm diameter guidance). In at leastone embodiment, the sheet 1902 may be sterilized with 0.1% (v/v)peracetic acid in PBS at 37° C. for 3 hours before being coated with themicrospheres 1604. The sheet 1902 is then spread/sprayed with a sealantor adhesive 1904 (such as fibrin sealant, for example). Fibrin sealanthas been applied for sealant or glue in various clinical practices andmay have positive effects on nerve regeneration.

Once the microspheres 1604 are loaded and axially aligned on the gratingmodule 1800 (either randomly or in a desired order), the sheet 1902 isapplied adhesive-side down to cover the grating module 1800 such thatthe microspheres 1604 affix to the sheet 1902 via the adhesive/sealant1904. After the adhesive/sealant 1904 is solidified or otherwise cured(if applicable), the sheet 1902 is separated from the grating module1800, thereby also removing the microspheres 1604 from the notches 1802of the grating module 1800 as well as they are now affixed to the sheet1902 via the adhesive/sealant 1904. Indeed, the resulting sheet 1902 iscoated with the microspheres 1604 aligned in one-by-one longitudinalrows thereon, forming the microchannels 1602 therebetween. Thetransverse dimensions of a single microchannel 1602 in the luminal graft10 is determined by the diameter of microspheres 1604 and interval 1804of the notch 1802 on the grating module 1800. Accordingly, the methoddescribed herein produces a sheet 2000 pre-coated with microspheres 1604that may then be used to fabricate the luminal graft 10.

Now referring to FIG. 20, one possible method for fabricating theluminal graft 10 from the pre-coated sheet 2000 is to roll the sheet2000 up into a multi-layer 12 cylinder. Fibrin sealant or anotheradhesive/sealant may be used to glue the layers 12 during rolling up thesheet 2000; however, the microspheres 1604 distributed within the layers12 will act as pillars and sustain the microchannels 1602 therein. Itwill be appreciated that, with this method for fabricating a luminalgraft 10, the initial size of the sheet 2000 will dictate the resultingdiameter of the luminal graft 10.

At least one alternative method for fabricating the luminal graft 10from the pre-coated sheet 2000 is shown in FIGS. 21A and 21B. There, aplurality of pre-coated sheets 2000 are stacked to assemble a cubicconstruct 2100 comprising multiple layers 12 (using fibrin sealant orother glues/adhesives between the layers 12). The precise number ofsheets 2000 used to form the cubic construct 2100 can be modifieddepending on the desired diameter of the resulting luminal graft 10. Inat least on embodiment, for example, the cubic construct 2100 maycomprise 5 to 20 sheets 2000 such that it comprises a thickness of 1 to4 mm.

A cylinder is trimmed from the cubic construct 2100 as shown in FIG.21B. In at least one embodiment, the cylinder is trimmed to comprise a 1to 4 mm diameter. Thereafter, an epineurium 2102 comprising a sheet ofPVP or other desired material (without microspheres 1604 affixedthereto) is wrapped around the trimmed cylinder and glued/sealed theretoto fabricate the luminal graft 10.

As previously noted, the fractional area of microchannels 1602 in theluminal graft 10 comprising microspheres 1604 is adjustable sincemicrosphere 1604 diameter and grating interval 1804 may vary arbitrarilyand the thickness of the tissue layer 12 (swine pulmonary ligamenttissue and/or visceral pleura in particular) can vary from 30 to 70 μm(depending on the regions harvested). Additionally, pulmonary ligamenttissue and/or visceral pleura thickness of animal species may vary fromabout 20 μm (rabbit) to about 300 μm (bovine). Accordingly, the broadrange of both microsphere 1604 dimensions and sheet thicknessesavailable provide the flexibility for optimization of fractional area ofthe microchannels 1602 present within the luminal graft 10.

In Vitro Studies

In vitro studies were performed to evaluate the composition andmechanical properties of fresh and fixed visceral pleura. Specifically,visceral pleura grafts 10 were implanted within murine 0.8 mm diameterfemoral arteries (n=6) for up to 6 months and canine 2.5 mm cerebralartery (n=2) for up to 2 months in order to address the safety andeffectiveness of visceral pleura as small-diameter vessel grafts.

In particular, the visceral pleura ultrastructure was viewed with amulti-photon microscope, with the representative fluorescent images areshown in FIG. 10A. The visceral pleura was found to be 64±6 mm thick.Furthermore, as illustrated in the bar graph of FIG. 10A, the visceralpleura was determined to contain about 11.9% collagen and about 13.2%elastin. Notably, the resulting C/E ratio of 1.10 for visceral pleurafalls within arteries 3.0-1.0 C/E ratio.

The biocompatibility of the visceral pleura was also assessed.Primarily, the visceral pleura was cut into 15 mm discs, placed intoindividual wells of a 24-well plate, and pretreated with 1 ml of fullmedia (DMEM supplemented with 10% FBS) for 24 hours at 37° C. prior totesting. Confluent NIH/3T3 fibroblasts were released from tissue cultureplates using 0.25% trypsin, spun, re-suspended, and counted. 100,000cells were added to each well in fresh media and incubated at 37° C. in5% CO₂ for 24 hours prior to imaging. In the final 2 hours prior toimaging, cells were exposed to Live/Dead (Invitrogen) pursuant to theassay instructions. Discs were subsequently imaged on a fluorescencemicroscope (Olympus) to determine cell attachment, and compared to smallintestine submucosa cells that had undergone like treatment. FIG. 10Cshows representative images of the visceral pleura (PVP) and smallintestine submucosa (SIS) following the study. Specifically, FIG. 10Chighlights reduced fibroblast adhesion to visceral pleura as compared tosmall intestine submucosa, thus supporting that visceral pleura tissueis indeed biocompatible.

In addition, cytotoxicity was evaluated in both 0.25% glutaraldehydefixed visceral pleura and small intestine submucosa tissue samples. Eachsample received a 10% v/v sterile filtered Fetal Bovine. Extracts wereprepared using trypsinized NIH-3T3 cells, which were placed into 6-wellplates in Full Media at a concentration of 1:40 v/v per well. The plateswere incubated overnight to promote attachment. The media was removedfrom each well and replaced with 2 mL of the appropriate extract. Theplates were then returned to the cell culture incubator and allowed anincubation time of 120 hours. Each well was imaged and rated fortoxicity.

FIG. 10D is representative of the results of this cytotoxicityevaluation, showing images of the visceral pleura (PVP) and smallintestine submucosa (SIS) tissue samples that illustrate reducedcytotoxicity of the visceral pleura tissue as compared to the smallintestine submucosa tissue. It is thought that the lower collagencontent of the visceral pleura as compared to other biologically testedscaffolds (pericardium and small intestine submucosa, for example)reduces cytotoxicity and, thus, would ultimately result in improvedclinical outcomes when used in grafting applications.

Finally, the mechanical properties of visceral pleura were evaluated.Such testing revealed that visceral pleura is more compliant thanpericardium. Furthermore, fresh versus fixed visceral pleura wereconfirmed to have similar mechanical properties, most likely due to thelarge elastin content within the tissue as elastin fibers do not undergofixation. In sum, the composition, biocompatibility, and materialproperties of the visceral pleura make it a strong candidate for vesselgrafts.

In Vivo Studies

A. Graft Performance in Vascular Applications

In addition to the previously described in vitro evaluations, in vivograft performance was also studied. Now referring to FIGS. 11A and 11B,schematic representations of the in vivo changes to a graft 10 followingimplantation are shown. In this at least one embodiment, the grafts 10evaluated comprised visceral pleura and were implanted in murine rightfemoral arteries (n=6, 0.80 mm in diameter) and canine internal carotidarteries (n=2, 2.5 mm in diameter). The femoral artery grafts 10 wereimplanted for 6 weeks, 12 weeks, and 6 months, while internal carotidartery grafts 10 were implanted for 2 months. Blood flow was assessed invivo in femoral grafts 10 using a transonic flow probe (TransonicSystems, Inc.), while carotid graft patency and flow were assessed usingan iE33 ultrasound system (Philips) Preliminary data showed that theblood flow in the right femoral grafts was not different as compared tocontrol left femoral arteries, indicative of a successful graftingprocedure. In addition, ultrasonic images revealed fully patent internalcarotid artery grafts.

On terminal procedure day, the grafts 10 were explanted and prepared forfluorescence imaging or functional assessment. Graft 10 tissue used forimmunofluorescence was sectioned using a cryotome and processed withprimary and secondary antibodies with fluorescence probes. Nuclei (FIG.11B-blue) were visualized with Hoechst 33442 (Life Technologies), whileelastin (FIG. 11B-red) was identified using anti-elastin antibody andvisualized with Alexa Fluor 546 (Life Technologies). Images wereobtained using a fluorescence microscope (TE300, Nikon). From thesefluorescence images, the full mechanism of the in vivo remodelingprocess in connection with the grafts 10 was studied.

Preliminary data from this in vivo study supports that the visceralpleura of the graft 10 initially acts as an internal elastic lamina andseparates endothelial cells and smooth muscle cells. As illustrated inFIG. 11A, at 10 days following implantation, there was a population ofsmooth muscle cells (SMC) on the abluminal side of the graft 10, with noevidence of endothelial cells (E) or an internal elastic lamina (IEL).At 12 weeks, the presence of internal elastic lamina was established, aswell as accompanying endothelial cells. At this point, smooth musclecells migrated within the graft 10 itself, which appeared to also beless dense—i.e. remodeled likely due to the matrix metalloproteinaseactivity of the myofibroblasts destined to be smooth muscle cells.Finally, at 6 months, the entire graft 10 matrix was occupied byorganized smooth muscle cells on the abluminal surface of the internalelastic lamina with endothelial cells on the luminal surface,characteristics indicative of a functional artery. Accordingly, theresulting functional organization was orchestrated by the host vesseland facilitated by the graft 10, with the two anastomotic ends being thesource of the migrating smooth muscle and endothelial cells and thegraft 10 guiding the proper migration thereof.

FIG. 11B shows immunofluorescence images of the graft 10 crosssection at10 days and 12 weeks following implantation (20 x objective). At 10days, the elastin fibers of the graft 10 can be clearly seen (see thearea highlighted by a dashed-white line, which displays as red in thecolorized version of the graph, indicative of the graft's 10 elastincontent), however smooth muscle cells have started to migrate on theabluminal side of the graft 10 (the remainder of the image whichdisplays as blue in the colorized version, indicative of the cellularnuclei). At 12 weeks post-implantation, the smooth muscle cells havesignificantly integrated into the graft 10, such that the areahighlighted by the dashed-white line shows up under immunofluorescenceas purple, thus confirming the increased distribution of smooth musclecells (blue under immunofluorescence) and the graft 10 (red underimmunofluorescence). The integration process of the smooth muscle cellsappears to be complete at 6 months post-implantation, with multiplelayers of organized smooth muscle cells.

In addition to the fluorescence imaging, isovolumic myography was usedto investigate vasoreactivity of the grafts 10. FIG. 12A shows arepresentative 0.80 mm diameter, visceral pleura graft 10 prior toimplantation and FIG. 12B shows a graphical representation of a visceralpleura graft's 10 functional response to pharmacological vasodilationand constriction after 6 months of in vivo remodeling. Specifically,Endothelin-1, a potent vasoconstrictor, was added to the perfusate toelicit graft constriction and the resulting intraluminal pressureincrease was measured. Conversely, the addition of acetylcholine (10⁻⁶M) resulted in endothelium-dependent vasodilation and a decrease inpressure. A further decrease in pressure was achieved by the addition ofsodium nitroprusside (10⁻⁵ M), which caused endothelium-independentvasodilation. As shown in FIG. 12B, the preliminary functional data fromthese studies supports that visceral pleura grafts 10 at 6 monthspost-implantation are responsive to pharmacological targeting ofendothelial and smooth muscle cells.

In sum, the overall data resulting from these in vivo studies isconsistent with the small-diameter (0.80-2.5 mm) visceral pleura grafts10 remodeling in vivo and ultimately developing into functional, patentconduits. Specifically, the data supports that the graft 10 facilitatesthe creation of a conduit that is structurally and functionallyequivalent to a native artery through use of the body's inherentphysiological remodeling process. Notably, these grafts 10 allmaintained their patency throughout this in vivo evaluation (6 months),even despite their notably small caliber (<1 mm in diameter). Thesefindings are of significant importance as they verify that the presentlydisclosed grafts 10 can provide a viable, and readily available, optionfor vessel grafts (including those of small-diameter) that can not onlyminimize vessel harvesting, reduce procedure times, and decrease theincidence of re-operations, but also significantly reduce patientmorbidity and mortality. In other words, the grafts 10 of the presentdisclosure can provide surgeons access to a variety of readilyavailable, efficacious, small- and large-diameter grafts, which willtranslate into improved clinical outcomes for a large patient populationand reduce overall healthcare costs.

B. Graft Performance in Nerve Conduit Applications

Additional in vivo graft performance was also studied in connection withnerve regeneration. Swine lungs were harvested from euthanized pigs,immediately stored in 4° C. saline, and the pulmonary visceral pleura(PVP) was gently separated from the lung. A solution containing 0.65%glutaraldehyde was used to crosslink the collagen, fibronectin, laminin,and glycocalyx of the PVP and the PVP was folded around a stainlesssteel module (˜1.6 mm diameter cylinder) and sealed longitudinally witha 10-0 suture to form a graft 10 for the sciatic nerve of rats. Thecylindrical grafts 10 were sterilized and rinsed 5 times by salinewithin 30 minutes before implantation in the murine sciatic neurotomy.The results supported that the conduit of PVP supplied by the graft 10successfully guided axonal regeneration, with the sciatic functionalindex of implanted PVP grafts 10 showing a significant improvement at12-weeks post-operation, indicating that sciatic nerve function wasimproved.

Additionally, immunofluorescence analysis indicated the successfulregeneration of nerve fibers through the PVP grafts 10. Perhaps morespecifically, biomarkers of peripheral nerve fiber were used to identifythe neo-generation of nerve fibers within the grafts 10. Neurofilament,a component of which is neurofilament light polypetide, is a majorcomponent of the neuronal cytoskeleton and functions primarily toimprove structural support for axon and to regulate axon diameter. Thepositive fluorescence images of neurofilament light polypeptide in thePVP graft 10 confirmed that the regeneration of axon bridges theproximal and distal stumps of the sciatic nerve. Indeed, the expressionof nerve growth factor receptors were strong in the neo-axon in the PVPgraft 10, and the extracellular matrices (laminin and sialic acid) foraxon growth and vasa vasorum also developed well.

To further validate the successful nerve regeneration using the PVPgraft 10, gastrocnemius muscle function was evaluated (gastrocnemiusmuscle function at least partially reflecting the function of thesciatic nerve). The gastrocnemius muscle in the murine leg innervated bythe sciatic nerve graft 10 was compared with the collateral legcomprising an intact sciatic nerve. The ratio of wet muscle mass was0.50±0.035, with the wet weight of the muscle in the graft legdecreasing to 2.6±0.8 gm (as compared to 6.5±1.3 gm in the control). Theimmunofluorescence images also indicated a decrease in the dimensions ofmuscular fibers, but sarcomere were of integration, which implicatespartial recovery of neuromuscular functions in the leg innervated by thenerve comprising the sciatic graft 10.

C. Graft Safety and Efficacy

In an additional study, visceral pleura grafts 10 were tested in achronic ischemic swine model to determine graft 10 safety (patency andthrombosis) and efficacy, as compared to a saphenous vein graft controlgroup. Graft 10 performance was evaluated in vivo using angiography,echocardiography, and electrocardiography. Grafts were also explanted tofurther assess graft 10 structure and function.

Primarily, lungs were obtained from a local abattoir and transported enbloc for processing. Within two (2) hours of explants, visceral pleurawas isolated via blunt dissection from the parenchyma taking care toavoid edges or fissures. Following isolation, specimens were rinsed andstored unstressed in 0.25% buffered glutaraldehyde solution at 23° C.for 48 hours. A sheet of visceral pleura was wrapped around a stainlesssteel cylinder and sutured together pursuant to the methods describedherein. The resulting grafts 10 were then rinsed with sterile salinefive times (5×) just before implantation surgery.

As swine coronary vasculature is more similar to humans as compared toother animal models, male swine were used in the in vivo studies (n=16,60-80 kg in weight). The swine were randomly divided into two groups,Group I and Group II. Two weeks prior to the bypass surgery, an ameroidconstrictor was placed around either the left anterior descendingartery, distal of the first diagonal, below the second diagonal, oraround the left circumflex artery to create an ischemia/infarct in eachswine to induce structural and functional heart changes. After two weeksof ameroid constriction, the swine underwent bypass surgery, with GroupI receiving the prepared pulmonary visceral pleura grafts 10, whileGroup II serving as a control group and receiving saphenous vein graftsas per conventional methodologies. Animals were then monitored over thenext 3 months, with angiography, echocardiography, electrocardiography,and blood sample collection done at baseline, 2, 4, 8, and 12 weeks.

On the day of non-survival surgery, all grafts were carefully excised,myocardial tissue collected for histology, and each heart was preservedfor tetrazolium chloride (TTC) staining Myocardial tissue samples(˜3×3×3 mm) were obtained from epicardium to endocardium and processedand embedded using conventional histological methods. Thin serialsections (˜2 μm) were cut and stained in order to visualize the bloodvessels and myocardium. The wall thickness of each graft was alsomeasured, including intima, media and adventitia.

Similarly, TTC staining was used to determine infarct size.Specifically, viable myocardium turned a deep red, while infractedtissue was white. Stained sections were then photographed and customMATLAB software was used to quantify the infarct size. Colored images ofTTC-stained sections were converted into an 8-bit scale and thresholdedto delineate infarct from viable tissue. Two-dimensional images werereconstructed to determine an accurate three-dimensional volume.

The vasoreactivity of the visceral pleura grafts 10, control saphenousvein grafts, and dissected circumflex artery were also determined usinga novel isovolumic myograph approach disclosed in U.S. PatentPublication Number 2009/0023176 to Kassab et al., which is incorporatedherein by reference. Generally, the vessels were each placed within asaline solution aerated with 95% O₂ and 5% CO₂ that was circulated in atissue bath and maintained at 37° C. Pharmacological agonists wereapplied externally to the vessel in the bath and the vessel'sintraluminal pressure was monitored during vessel contraction andexpansion. Additionally, changes in vessel shape were also recorded witha CCD camera.

In line with other studies, both the visceral pleura grafts 10 and thesaphenous vein grafts reversed ischemic damage in each of the respectivehearts, including improving electrocardiographic and echocardiographicparameters. Additionally, the visceral pleura grafts 10 avoided dilationand exhibited improved patency as compared to the saphenous vein grafts(controls) at 3 months post-implantation. As supported by the results ofboth the in vitro and in vivo evaluations, visceral pleura—and liketissue having a high elastin content and mesothelial cells such aspulmonary ligament tissue—has significant potential for use in graftingapplications and, in particular, luminal grafts with a small-diameter.Due to the availability of such tissue, the use of these tissues inluminal grafting and, specifically, CABG surgery could address asignificant clinical problem as it would provide surgeons with numerousconduit size options even with respect to small-diameter grafts.

Additionally, the biocompatibility of swine PVP implanted as bloodvessel (patch and graft) and skin grafts was also evaluated. In 4 pigs,carotid arteries remained 100% patency and arterial incisions wereremodeled to similar medial thickness with phenotype vascular smoothmuscle cells 3 months post-operation following the implantation of swinePVP as carotid artery patches (7 mm×15 mm). Similarly, jugular veins in6 pigs all remained patency following the implantation of swine PVP asjugular vein patches (12 mm×32 mm), with the incisions under the patchessuccessfully remolding the similar tissue to native vein. Swine PVPimplanted in the femoral arteries (0.7 mm luminal diameter) of 30 ratsalso exhibited very high patency of 91% 3 months post-operation,indicative of excellent biocompatibility. These findings indicate swinePVP in particular can provide a beneficial biomaterial for nerveguidance conduit as well as in vascular applications.

The various devices, systems, and methods for replacing damaged orcompromised blood vessels and engineering luminal grafts for variousmedical applications have various benefits to patients in need ofvascular surgery or nerve regeneration. For example, the devices,systems and methods hereof may be employed to facilitate vesselreplacement for patients in need of replacement blood vessels including,without limitation, small-diameter conduit vessels. Furthermore, thedevices, systems and methods of the present disclosure may be employedto facilitate placement of vascular access—namely, an arteriovenousgraft—in connection with delivery of hemodialysis. Additionally, thedevices, systems and methods herein may also be employed in connectionwith nerve regeneration applications and, in particular, tubulizationand nerve guidance conduits. FIGS. 14A and 14B show A) a luminal graftaccording to the present disclosure being used as a nerve guidanceconduit, and B) the end of the nerve guidance conduit of FIG. 14B. FIG.14C shows a close-up view of a sample of pulmonary pleura in vivo.

We have previously disclosed pulmonary visceral pleura (PVP) andpulmonary ligament (PL) as novel tissue for graft purposes, such asdisclosed within U.S. patent application publication nos. 2015/0064140and 2006/0067031 of Kassab et al. To date, our experience show excellentpatency (greater 90%) in as long as nine months in a rat model and theformation of a neo-artery (0.8 mm in diameter) from the graft withfunctional endothelium and smooth muscle cells. In addition to theseexcellent findings, the AVG requires resistance to multiple (3-4) weeklypunctures for dialysis. Since the native PVP and PL tissues are too thinand the need for maturation (thickening of wall) would take severalmonths, it is desirable to combine the ideal surface of PVP or PL with asynthetic tube material (i.e., biological/synthetic hybrid) ofappropriate thickness that can withstand multiple punctures with largebore needle. There are at least two approaches to accomplishing thedesired outcome.

One exemplary approach is depicted in FIG. 22 and FIG. 23. As showntherein, an exemplary graft 10 (showing inner lumen 14, multiple layers12 (noting that an exemplary graft 10 referenced herein can have one ormore layers 12, such as the one layer 12 shown in FIG. 1 and themultiple layers 12 shown in FIG. 2), inner surfaces 17 of layers 12, andouter surfaces 19 of layers 12) utilizes one or more synthetic materials2200 (silicone, PTFE, elastomer, or other biologically compatiblematerials) as one or more layers 12 of graft 10 material, and at leastone inner surface 17, such as the inner surface 17 of the layer 12 withthe ultimate graft 10 lumen 14) is lined with a biological material 2202(shown as a series of diagonal lines in FIG. 22), such as PVP or PL,using one or more biological glues 2204 (UV cured, etc.) or othermechanical fixation of the biological material 2202 to the inner surface17 of the graft 10 (also referred to in such embodiments as a syntheticgraft 10, as it comprises synthetic material(s) 2200 and biologicalmaterial(s) 2202). FIG. 23 shows a graft 10 having one layer 12, asopposed to the multiple layer 12 graft 10 shown in FIG. 22, whereby abiological glue 2204 is shown as being used/applied to help affixbiological material 2202 to inner surface 17 of layer 12. FIG. 24 showsa graft 10 also having one layer 12, whereby the biological material isapplied directly to inner surface 17 of layer 12, such as either notrequiring a biological glue 2204 or incorporating a biological glue 2204with biological material 2202 to help with application/adhesion.

Another exemplary approach, for example, is to harvest the mesotheliumand other surface molecules (glycocalyx such as hyaluronic acid, heparansulfate, sialic acid, etc.) of the PVP and PL (also referred to hereinas biological material 2202) such that they (the biological material2202) can be sprayed onto the inner surface 17 of a layer 12 of thehybrid graft 10 in combination with adhesion molecules (e.g., platelets)(also referred to herein a biological glues). As such, FIGS. 22 and 23also depict such an embodiment, whereby biological material 2202 can beapplied to inner surface 17 of a layer 12 of the hybrid graft 10, suchas by spraying, and an optional biological glue 2204 can also be usedwhereby said biological glue 2204 is applied, such as by spraying orother application method, prior to or during application of biologicalmaterial 2202. Biological material 2202 and biological glue 2204 canalso be combined and applied to inner surface 17 of a layer 12 of thehybrid graft 10 at a single time, such as by spraying.

FIG. 24 shows a portion of an exemplary hybrid graft 10 of the presentdisclosure. As shown therein, hybrid graft 10 comprises a syntheticmaterial 2200 and a biological material 2202, whereby the syntheticmaterial 2200 and the biological material 2202 are generally configuredas layers. In various embodiments, synthetic material 2200 may be acontiguous layer of material, and biological material 2202 may be acomplete/contiguous layer of material or less than a complete/contiguouslayer of material. Hybrid graft 10, as shown in FIG. 24, could be shapedas desired, such as ultimately shaped as a cylinder or tube.

FIG. 25 shows an exemplary hybrid graft 10 of the present disclosureconfigured as a cylinder or tube. As shown therein (in cross-section),hybrid graft 10 comprises a synthetic material 2200 on a relativeoutside and a biological material 2202 on a relative inside, such thatlumen 14 (defined by one or both of synthetic material 2200 and/orbiological material 2202) can allow fluid flow therethrough, such asblood, when implanted into a mammalian body. In such a configuration,the fluid (such as blood) would contact the biological material 2202 ofhybrid graft 10, while the synthetic material 2200 would be on arelative outside, such as contacting a wall of a blood vessel.

FIG. 26 shows an exemplary hybrid graft 10 of the present disclosureconfigured as a cylinder or tube. As shown therein (in cross-section),hybrid graft 10 comprises a biological material 2202 on a relativeoutside, a synthetic material 2200 on a relative inside of thatbiological material 2202 layer, and another biological material 2202 ona relative inside, such that lumen 14 (defined by one or both ofsynthetic material 2200 and/or biological material 2202) can allow fluidflow therethrough, such as blood, when implanted into a mammalian body.In such a configuration, the fluid (such as blood) would contact theinner layer of biological material 2202 of hybrid graft 10, while theouter layer of biological material 2202 would be on a relative outside,such as contacting a wall of a blood vessel, such that the syntheticmaterial 2200 may not contact the wall of the blood vessel or fluidflowing through the graft 10.

In various embodiments, synthetic material 2200 comprises a layer ofmaterial, and biological material 2202 is applied thereto, either as itsown separate layer physically applied thereto, or sprayed thereon, suchas by way of a mixture of biological material 2202 and another material,whereby the mixture as a consistency that can be applied to the layer ofsynthetic material 2200. For example, and as shown in FIG. 27, a mixture2700 of the present disclosure can comprise biological material 2202, asreferenced, and at least one additional material 2702, such as a liquid(water, saline, blood, etc.), a biological glue 2204, or anothermaterial.

The novel biological/synthetic hybrid graft 10 of the present disclosureoffers a new vascular access paradigm where long access life, lowmaintenance, and extended interventional intervals are the design goals.This hybrid approach should demonstrate promising performancecharacteristics such as, antithrombogenesis, a highly mobile structuresupporting mechanical arterial behavior and possibly the ability to sealreadily following large needle puncture, high resistance to neointimalhyperplasia, and arterial function propagation within the graft 10 bythe host. The hybrid graft 10 is especially suited for low flowenvironments, small diameter applications, and other hostile implantenvironments which makes it unique within the current graft offerings.These tissue characteristics match up very well to many of the majorconcerns in the current hemodialysis environment and may offercompelling short and long-term outcomes.

The impact and benefits of the tissue-engineered graft 10 are numerousand significant. Primarily, the hybrid graft 10 can be characterized bythe preferred properties of both the fistula and graft, and at the sametime, minimize the negatives of both solutions. This novel hybrid graft10 implant can mature in the near-term like a fully biological graft 10of the present disclosure, but in the long-term shows traits similar tofistulas. This hybrid graft 10 can demonstrate several importantadvancements that ultimately support longer access site life for ESRDpatients on hemodialysis and the physician's maintenance efforts. First,the hybrid graft 10 solution can offer a readily implantable andpredictable platform for surgeons to create vascular access, fastimplant-to-first dialysis times, and low failure-to-mature rates thatare synonymous with grafts. This hybrid graft 10 offers naturalantithrombogenesis performance that lowers the potential for thrombusformation, seen commonly in fistulas and grafts. The hybrid graft 10 canshow a unique property in which the host integrates the graft 10 into anartery-like state possibly translating to the long-term benefits of anative fistula (low infection rates and long life). Due to itsglycocalyx content, it demonstrates a high resistance to neointimalhyperplasia, which is common in fistulas due to the repeat needleinjury, leading to lower stenotic occurrences within the graft 10. Onedistinct benefit with the availability of this graft 10 would be fewerlonger-term tunneled catheter (TC) dialysis patients. In the situationwhere TC use cannot be avoided and our graft 10 is implanted, the lengthof time where the TC would likely be short because it's a graft 10. Froma cost expenditure perspective, an exemplary hybrid graft 10 of thepresent disclosure takes less time and processing to manufacture as seenin other engineered grafts, namely it does not require smooth musclecell or fibroblast seeding and ingrowth, yielding a lower cost ofproduction. For hemodialysis dependent patients, the uniquecharacteristics of this novel graft 10 stands to make a significantcontribution in extending the life of a vascular access site andlowering the number of interventions required to maintain access sitepatency.

While embodiments of grafts and methods of making and using the samehave been described in considerable detail herein, the embodiments aremerely offered by way of non-limiting examples of the disclosuredescribed herein. It will therefore be understood that various changesand modifications may be made, and equivalents may be substituted forelements thereof, without departing from the scope of the disclosure.Indeed, this disclosure is not intended to be exhaustive or to limit thescope of the disclosure.

Further, in describing representative embodiments, the disclosure mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described.Other sequences of steps may be possible. Therefore, the particularorder of the steps disclosed herein should not be construed aslimitations of the present disclosure. In addition, disclosure directedto a method and/or process should not be limited to the performance oftheir steps in the order written. Such sequences may be varied and stillremain within the scope of the present disclosure.

1. A hybrid luminal graft comprising a generally tubular element, theluminal graft comprising: a first layer comprising one or more syntheticmaterials, the first layer defining an inner surface and an opposingouter surface; a biological material applied to the inner surface of thefirst layer, the biological tissue having elastin fibers and collagenfibers, with the elastin fibers being a dominant component thereof, andwherein the biological material is selected from the group consisting ofpulmonary visceral pleura, pulmonary ligament, a component harvestedfrom pulmonary visceral pleura, and a component harvested from pulmonaryligament; and a plurality of microchannels formed on a surface of thebiological material, each of the microchannels extending longitudinallybetween a first end and a second end of the biological material andconfigured to provide intraluminal structural guidance to nerve cellsproliferating therethrough; wherein when the first layer is configuredas a generally tubular element, the biological material is presentwithin a defined lumen of the first layer.
 2. The hybrid luminal graftof claim 1, wherein the synthetic material is silicone.
 3. The hybridluminal graft of claim 1, wherein the synthetic material ispolytetrafluoroethylene.
 4. The hybrid luminal graft of claim 1, whereinthe synthetic material is elastomer.
 5. The hybrid luminal graft ofclaim 1, wherein a biological glue is used to facilitate adherence ofthe biological material to the inner surface.
 6. The hybrid luminalgraft of claim 1, wherein the biological material comprises glycocalyx.7. The hybrid luminal graft of claim 1, further comprising: a secondlayer comprising one or more synthetic materials, the second layerpositioned around a relative outside of the first layer.
 8. The hybridluminal graft of claim 7, further comprising: a third layer comprisingone or more synthetic materials, the third layer positioned around arelative outside of the second layer.